Compositions and methods for regulating glucose metabolism

ABSTRACT

The present disclosure provides compositions for regulating glucose metabolism. The compositions provide for reduced levels of p75 NTR  and/or reduced binding of p75 NTR  to a GTPase such as Rab31 or Rab5. The compositions are useful in methods of regulating glucose metabolism, which methods are also provided.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional PatentApplication No. 61/580,523, filed Dec. 27, 2011, which application isincorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under Grant No. NS51470awarded by the National Institutes of Health. The government has certainrights in the invention.

BACKGROUND

Insulin resistance is a key feature of type 2 diabetes, and ischaracterized by decreased glucose disposal, increased hepatic glucoseproduction, with a reduced ability of insulin to maintain normal glucosehomeostasis. Whole-body glucose metabolism is regulated by a complexcommunication network between various tissues, including adipose tissue,liver, muscle, and brain. Glucose transporter-4 (GLUT4) is the principalinsulin-stimulated glucose transporter expressed primarily in adiposetissue and skeletal muscle. Insulin stimulates glucose uptake byinducing GLUT4 translocation to the plasma membrane. GLUT4 traffickingfrom intracellular compartments to the plasma membrane is regulated by anumber of small GTPases, including Rab5 and its family member Rab31.

p75^(NTR) is a member of the Tumor Necrosis Factor (TNF) receptorsuperfamily that was originally identified as a receptor forneurotrophins p75^(NTR) is expressed in the nervous system and in adultnon-neuronal tissues, such as white adipose tissue (WAT), and muscle.p75^(NTR) has surprisingly diverse cellular functions, includingmodulation of cell survival, apoptosis and differentiation, whichunderlie its in vivo biologic functions, including liver and muscleregeneration, extracellular matrix remodeling, sensory neurondevelopment, hypoxia, and the angiogenic response.

Literature

Dugani and Klip (2005) EMBO Rep 6:1137-1142; Lodhi et al. (2007) CellMetab 5:59-72; Peeraully et al. (2004) Am J Physiol Endocrinol Metab287:E331-339; Sachs et al. (2007) J Cell Biol 177:1119-1132; Liebinsh etal. (1997) EMBO J. 16:4999; Yamashita and Tohyama (2003) Nat Neurosci6:461-467

SUMMARY

The present disclosure provides compositions for regulating glucosemetabolism. The compositions provide for reduced levels of p75^(NTR)and/or reduced binding of p75^(NTR) to a GTPase such as Rab31 or Rab5.The compositions are useful in methods of regulating glucose metabolism,which methods are also provided.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-H depict features of p75^(NTR−/−) mice.

FIGS. 2A-C depict metabolic analysis of p75^(NTR−/−) mice on normalchow.

FIGS. 3A-D depict basal levels of free fatty acids (FFA) (A), bloodglucose (B), and insulin (C) after claim experiments in wild-type (WT)and p75^(NTR−/−) mice; FIG. 3D depicts western blot analysis forp75^(NTR) in mouse embryonic fibroblast (MEF)-derived adipocytes.

FIGS. 4A-E depict regulation of glucose uptake by p75^(NTR) inadipocytes and skeletal muscle cells.

FIGS. 5A and 5B depict basal and insulin-stimulated glucose uptake in3T3L1 adipocytes.

FIGS. 6A and 6B depict regulation of GLUT4 trafficking in adipocytes byp75^(NTR).

FIGS. 7A-D depict western blots for Glut1, Glut4, and p75^(NTR) inadipocytes.

FIGS. 8A and 8B depict regulation of Rab5 and Rab31 GTPase activity byp75^(NTR) in adipocytes.

FIGS. 9A-I depict interaction of the death domain of p75^(NTR) with Rab5and Rab31 GTPases. FIG. 9H depicts Peptide 2: NSRPVNQPRPEGEK (SEQ IDNO:33); Peptide 4: GQALKGDGGLYSSLP (SEQ ID NO:34); and Peptide 7:LLASWATQDSATLDA (SEQ ID NO:1).

FIGS. 10A-D depict aspects of the interaction of p75^(NTR) with Rab5 andRab31 GTPases. FIG. 10 D depicts the peptideTHEACPVRALLASWATQDSATLDALLAALRRIQRA (SEQ ID NO:35).

FIG. 11 provides an amino acid sequence of a p75^(NTR) polypeptide (SEQID NO:45)

FIG. 12 provides an amino acid sequence alignment of the death domain ofp75^(NTR) of various species. Human (SEQ ID NO:46); Chimp (SEQ IDNO:47); Dog (SEQ ID NO:48); Rat (SEQ ID NO:49); Mouse (SEQ ID NO:50).

FIG. 13 provides an amino acid sequence of a Rab5 GTPase (SEQ ID NO:51).

FIG. 14 provides an amino acid sequence of a Rab31 GTPase (SEQ IDNO:52).

FIG. 15 provides a nucleotide sequence encoding a p75^(NTR) polypeptide(SEQ ID NO:53).

FIG. 16 depicts peptide array data of Rab31 peptides with p75^(NTR)intracellular domain (ICD). Rab31 (SEQ ID NO:54); Rab31 Peptide 2 (SEQID NO:55); Rab31 Peptide 6 (SEQ ID NO:17).

DEFINITIONS

The phrase “glucose tolerance”, as used herein, refers to the ability ofa subject to control the level of plasma glucose and/or plasma insulinwhen glucose intake fluctuates. For example, glucose toleranceencompasses the ability to reduce the level of plasma glucose back to alevel before the intake of glucose within about 120 minutes or so.

The phrase “pre-diabetes”, as used herein, refers a condition that maybe determined using either the fasting plasma glucose test (FPG) or theoral glucose tolerance test (OGTT). Both require a person to fastovernight. In the FPG test, a person's blood glucose is measured firstthing in the morning before eating. In the OGTT, a person's bloodglucose is checked after fasting and again 2 hours after drinking aglucose-rich drink. In a healthy individual, a normal test result of FPGwould indicate a glucose level of below about 100 mg/dl. A subject withpre-diabetes would have a FPG level between about 100 and about 125mg/dl. If the blood glucose level rises to about 126 mg/dl or above, thesubject is determined to have “diabetes”. In the OGTT, the subject'sblood glucose is measured after a fast and 2 hours after drinking aglucose-rich beverage. Normal blood glucose in a healthy individual isbelow about 140 mg/dl 2 hours after the drink. In a pre-diabeticsubject, the 2-hour blood glucose is about 140 to about 199 mg/dl. Ifthe 2-hour blood glucose rises to 200 mg/dl or above, the subject isdetermined to have “diabetes”.

The term “Rab5 GTPase” refers to a polypeptide having GTPase activity,and sharing at least about 75%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity with a contiguous stretch of from about 150amino acids to about 200 amino acids, or from about 200 amino acids toabout 215 amino acids, of the amino acid sequence depicted in FIG. 13(SEQ ID NO:51). See also GenBank Accession No. AAB08927; and GenBankAccession No. NP_080163. The crystal structure of human Rab5 has beenreported; see, e.g., Zhu et al. (2003) J. Biol. Chem. 278:2452.

The term “Rab31 GTPase” refers to a polypeptide having GTPase activity,and sharing at least about 75%, at least about 85%, at least about 90%,at least about 95%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity with a contiguous stretch of from about 125amino acids to about 150 amino acids, from about 150 amino acids toabout 175 amino acids, or from about 175 amino acids to about 195 aminoacids, with the amino acid sequence depicted in FIG. 14 (SEQ ID NO:52).See also GenBank Accession No. CAG38587; and GenBank Accession No.NP_598446; and Bao et al. (2002) Eur. J. Biochem. 269:259.

Before the present invention is further described, it is to beunderstood that this invention is not limited to particular embodimentsdescribed, as such may, of course, vary. It is also to be understoodthat the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting, sincethe scope of the present invention will be limited only by the appendedclaims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is encompassed within the invention. The upper and lowerlimits of these smaller ranges may independently be included in thesmaller ranges, and are also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described. All publications mentionedherein are incorporated herein by reference to disclose and describe themethods and/or materials in connection with which the publications arecited.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural referents unless thecontext clearly dictates otherwise. Thus, for example, reference to “ap75^(NTR)-derived peptide” includes a plurality of such peptides andreference to “the p75^(NTR)-specific shRNA” includes reference to one ormore p75^(NTR)-specific shRNAs and equivalents thereof known to thoseskilled in the art, and so forth. It is further noted that the claimsmay be drafted to exclude any optional element. As such, this statementis intended to serve as antecedent basis for use of such exclusiveterminology as “solely,” “only” and the like in connection with therecitation of claim elements, or use of a “negative” limitation.

It is appreciated that certain features of the invention, which are, forclarity, described in the context of separate embodiments, may also beprovided in combination in a single embodiment. Conversely, variousfeatures of the invention, which are, for brevity, described in thecontext of a single embodiment, may also be provided separately or inany suitable sub-combination. All combinations of the embodimentspertaining to the invention are specifically embraced by the presentinvention and are disclosed herein just as if each and every combinationwas individually and explicitly disclosed. In addition, allsub-combinations of the various embodiments and elements thereof arealso specifically embraced by the present invention and are disclosedherein just as if each and every such sub-combination was individuallyand explicitly disclosed herein.

The publications discussed herein are provided solely for theirdisclosure prior to the filing date of the present application. Nothingherein is to be construed as an admission that the present invention isnot entitled to antedate such publication by virtue of prior invention.Further, the dates of publication provided may be different from theactual publication dates which may need to be independently confirmed.

DETAILED DESCRIPTION

The present disclosure provides compositions for regulating glucosemetabolism. The compositions provide for reduced levels of p75^(NTR)and/or reduced binding of p75^(NTR) to a GTPase such as Rab31 or Rab5.The compositions are useful in methods of regulating glucose metabolism,which methods are also provided.

p75^(NTR)-Derived Peptides

The present disclosure provides p75^(NTR)-derived peptides that exhibitone or more of the following activities: 1) reduces binding offull-length p75^(NTR) to Rab5 GTPase; 2) reduces binding of full-lengthp75^(NTR) to Rab31 GTPase; 3) increases glucose disposal rate (GDR); 4)increases the insulin-stimulated GDR; 5) increases insulin-stimulateduptake of glucose into adipocytes and myocytes; 6) increases GLUT4trafficking, e.g., increases GLUT4 translocation to the plasma membrane,e.g., in adipocytes and myocytes; 7) increases insulin sensitivity.

In some cases, a p75^(NTR)-derived peptide enhances glucose uptake intoa cell, e.g., in some cases, a p75^(NTR)-derived peptide increasesinsulin-stimulated uptake of glucose into an adipocyte and/or a myocyte.For example, in some embodiments, a subject p75^(NTR)-derived peptideincreases insulin-stimulated uptake of glucose into an adipocyte and/ora myocyte by at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 50%, at least about 75%, at leastabout 2-fold, at least about 5-fold, at least about 10-fold, or morethan 10-fold, compared to the insulin-stimulated uptake of glucose intothe adipocyte or myocyte in the absence of the p75^(NTR)-derivedpeptide.

In some instances, a subject p75^(NTR)-derived peptide can increaseglucose uptake into an adipocyte and/or a myocyte with an EC₅₀ in therange of from about 0.0001 nM to 100 μM (e.g., from about 0.0001 nM toabout 0.001 nM, from about 0.001 nM to about 0.01 nM, from about 0.01 nMto about 0.1 nM, from about 0.1 nM to about 1 nM, from about 1 nM toabout 10 nM, from about 10 nM to about 100 nM, from about 100 nM toabout 1 μM, from about 1 μM to about 10 μM, or from about 10 μM to about100 μM) in an in vitro assay for glucose uptake.

In some cases, a p75^(NTR)-derived peptide has a length of from about 15amino acids to about 20 amino acids, from about 20 amino acids to about25 amino acids, from about 25 amino acids to about 30 amino acids, fromabout 30 amino acids to about 35 amino acids, from about 35 amino acidsto about 40 amino acids, from about 40 amino acids to about 45 aminoacids, from about 45 amino acids to about 50 amino acids, from about 50amino acids to about 55 amino acids, from about 55 amino acids to about60 amino acids, from about 60 amino acids to about 65 amino acids, fromabout 65 amino acids to about 70 amino acids, from about 70 amino acidsto about 75 amino acids, from about 75 amino acids to about 80 aminoacids, from about 80 amino acids to about 85 amino acids, from about 85amino acids to about 90 amino acids, from about 90 amino acids to about95 amino acids, or from about 95 amino acids to about 100 amino acids.

In some embodiments, a p75^(NTR)-derived peptide has a length of fromabout 15 amino acids to about 100 amino acids, as set out above, and hasat least about 90%, at least about 95%, at least about 97%, at leastabout 98%, at least about 99%, or 100%, amino acid sequence identitywith a contiguous stretch of from about 15 amino acids to about 20 aminoacids, from about 20 amino acids to about 25 amino acids, from about 25amino acids to about 30 amino acids, from about 30 amino acids to about35 amino acids, from about 35 amino acids to about 40 amino acids, fromabout 40 amino acids to about 45 amino acids, from about 45 amino acidsto about 50 amino acids, from about 50 amino acids to about 55 aminoacids, from about 55 amino acids to about 60 amino acids, from about 60amino acids to about 65 amino acids, from about 65 amino acids to about70 amino acids, from about 70 amino acids to about 75 amino acids, fromabout 75 amino acids to about 80 amino acids, from about 80 amino acidsto about 85 amino acids, from about 85 amino acids to about 90 aminoacids, or from about 90 amino acids to 92 amino acids or 95 amino acids,of an amino acid sequence depicted in FIG. 12, i.e., one of SEQ IDNOs:46-50.

In some cases, a p75^(NTR)-derived peptide comprises one of thefollowing amino acid sequences:

a) LLASWATQDSATLDA (SEQ ID NO:1);

b) X₁LASWATQDSATLDA (SEQ ID NO:2);

c) LX₁ASWATQDSATLDA (SEQ ID NO:3);

d) LLX₁SWATQDSATLDA (SEQ ID NO:4);

e) LLAX₁WATQDSATLDA (SEQ ID NO:5);

f) LLASX₁ATQDSATLDA (SEQ ID NO:6);

g) LLASWX₁TQDSATLDA (SEQ ID NO:7);

h) LLASWAX₁QDSATLDA (SEQ ID NO:8);

i) LLASWATX₁DSATLDA (SEQ ID NO:9);

j) LLASWATQX₁SATLDA (SEQ ID NO:10);

k) LLASWATQDX₁ATLDA (SEQ ID NO: 11);

l) LLASWATQDSX₁TLDA (SEQ ID NO:12);

m) LLASWATQDSAX₁LDA (SEQ ID NO:13);

n) LLASWATQDSATX₁DA (SEQ ID NO:14);

o) LLASWATQDSATLX₁A (SEQ ID NO:15); and

p) LLASWATQDSATLDX₁ (SEQ ID NO:16),

where the peptide has a length of from about 15 amino acids to about 20amino acids, from about 20 amino acids to about 25 amino acids, fromabout 25 amino acids to about 30 amino acids, from about 30 amino acidsto about 35 amino acids, from about 35 amino acids to about 40 aminoacids, from about 40 amino acids to about 45 amino acids, from about 45amino acids to about 50 amino acids, or from about 50 amino acids toabout 100 amino acids; and where X₁ is any amino acid.Rab31-Derived Peptides

The present disclosure provides Rab31-derived peptides that exhibit oneor more of the following activities: 1) reduces binding of full-lengthp75^(NTR) to Rab31 GTPase; 2) increases glucose disposal rate (GDR); 3)increases the insulin-stimulated GDR; 4) increases insulin-stimulateduptake of glucose into adipocytes and myocytes; 5) increases GLUT4trafficking, e.g., increases GLUT4 translocation to the plasma membrane,e.g., in adipocytes and myocytes; 6) increases insulin sensitivity.

In some cases, a Rab31-derived peptide enhances glucose uptake into acell, e.g., in some cases, a Rab31-derived peptide increasesinsulin-stimulated uptake of glucose into an adipocyte and/or a myocyte.For example, in some embodiments, a subject Rab31-derived peptideincreases insulin-stimulated uptake of glucose into an adipocyte and/ora myocyte by at least about 15%, at least about 20%, at least about 25%,at least about 30%, at least about 50%, at least about 75%, at leastabout 2-fold, at least about 5-fold, at least about 10-fold, or morethan 10-fold, compared to the insulin-stimulated uptake of glucose intothe adipocyte or myocyte in the absence of the Rab31-derived peptide.

In some instances, a subject Rab31-derived peptide can increase glucoseuptake into an adipocyte and/or a myocyte with an EC₅₀ in the range offrom about 0.0001 nM to 100 μM (e.g., from about 0.0001 nM to about0.001 nM, from about 0.001 nM to about 0.01 nM, from about 0.01 nM toabout 0.1 nM, from about 0.1 nM to about 1 nM, from about 1 nM to about10 nM, from about 10 nM to about 100 nM, from about 100 nM to about 1μM, from about 1 μM to about 10 μM, or from about 10 μM to about 100 μM)in an in vitro assay for glucose uptake.

In some cases, a Rab31-derived peptide comprises one of the followingamino acid sequences:

EHGPENIVMAIAGNK; (SEQ ID NO: 17) EX₁GPENIVMAIAGNK; (SEQ ID NO: 18)EHX₁PENIVMAIAGNK; (SEQ ID NO: 19) EHGX₁ENIVMAIAGNK; (SEQ ID NO: 20)EHGPX₁NIVMAIAGNK; (SEQ ID NO: 21) EHGPX₁NIVMAIAGNK; (SEQ ID NO: 22)EHGPEX₁IVMAIAGNK; (SEQ ID NO: 23) EHGPENX₁VMAIAGNK; (SEQ ID NO: 24)EHGPENIX₁MAIAGNK; (SEQ ID NO: 25) EHGPENIVX₁AIAGNK; (SEQ ID NO: 26)EHGPENIVMX₁IAGNK; (SEQ ID NO: 27) EHGPENIVMAX₁AGNK; (SEQ ID NO: 28)EHGPENIVMAIX₁GNK; (SEQ ID NO: 29) EHGPENIVMAIAX₁NK; (SEQ ID NO: 30)EHGPENIVMAIAGX₁K;  (SEQ ID NO: 31) and EHGPENIVMAIAGNX₁, (SEQ ID NO: 32)

where the peptide has a length of from about 15 amino acids to about 20amino acids, from about 20 amino acids to about 25 amino acids, fromabout 25 amino acids to about 30 amino acids, from about 30 amino acidsto about 35 amino acids, from about 35 amino acids to about 40 aminoacids, from about 40 amino acids to about 45 amino acids, from about 45amino acids to about 50 amino acids, or from about 50 amino acids toabout 100 amino acids; and where X₁ is any amino acid.

Amide Bond Substitutions

In some cases, a peptide of the present disclosure (e.g., ap75^(NTR)-derived peptide; a Rab31-derived peptide) includes one or morelinkages other than peptide bonds, e.g., at least two adjacent aminoacids are joined via a linkage other than an amide bond. For example, toreduce or eliminate undesired proteolysis or other degradation pathwaysand/or to increase serum stability and/or to restrict or increaseconformational flexibility, one or more amide bonds within the backboneof a p75^(NTR)-derived peptide or a Rab31-derived peptide can besubstituted.

For example, one or more amide linkages (—CO—NH—) in a p75^(NTR)-derivedpeptide or a Rab31-derived peptide can be replaced with another linkagewhich is an isostere such as: —CH₂NH—, CH₂S—, —CH₂CH₂—, —CH═CH— (cis andtrans), —COCH₂—, —CH(OH)CH₂— and —CH₂SO—. This replacement can be madeby methods known in the art.

As another example, one or more amide linkages in a p75^(NTR)-derivedpeptide or a Rab31-derived peptide can be replaced with a reducedisostere pseudopeptide bond. Couder et al. (1993) Int. J. PeptideProtein Res. 41:181-184.

Amino Acid Substitutions

One or more amino acid substitutions can be made in a p75^(NTR)-derivedpeptide or a Rab31-derived peptide. The following are non-limitingexamples:

-   -   a) substitution of alkyl-substituted hydrophobic amino acids:        including alanine, leucine, isoleucine, valine, norleucine,        (S)-2-aminobutyric acid, (S)-cyclohexylalanine or other simple        alpha-amino acids substituted by an aliphatic side chain from        C₁-C₁₀ carbons including branched, cyclic and straight chain        alkyl, alkenyl or alkynyl substitutions;    -   b) substitution of aromatic-substituted hydrophobic amino acids:        including phenylalanine, tryptophan, tyrosine, sulfotyrosine,        biphenylalanine, 1-naphthylalanine, 2-naphthylalanine,        2-benzothienylalanine, 3-benzothienylalanine, histidine,        including amino, alkylamino, dialkylamino, aza, halogenated        (fluoro, chloro, bromo, or iodo) or alkoxy (from        C₁-C₄)-substituted forms of the above-listed aromatic amino        acids, illustrative examples of which are: 2-, 3- or        4-aminophenylalanine, 2-, 3- or 4-chlorophenylalanine, 2-, 3- or        4-methylphenylalanine, 2-, 3- or 4-methoxyphenylalanine,        5-amino-, 5-chloro-, 5-methyl- or 5-methoxytryptophan, 2′-, 3′-,        or 4′-amino-, 2′-, 3′-, or 4′-chloro-, 2, 3, or        4-biphenylalanine, 2′-, 3′-, or 4′-methyl-, 2-, 3- or        4-biphenylalanine, and 2- or 3-pyridylalanine;    -   c) substitution of amino acids containing basic side chains:        including arginine, lysine, histidine, ornithine,        2,3-diaminopropionic acid, homoarginine, including alkyl,        alkenyl, or aryl-substituted (from C₁-C₁₀ branched, linear, or        cyclic) derivatives of the previous amino acids, whether the        substituent is on the heteroatoms (such as the alpha nitrogen,        or the distal nitrogen or nitrogens, or on the alpha carbon, in        the pro-R position for example. Compounds that serve as        illustrative examples include: N-epsilon-isopropyl-lysine,        3-(4-tetrahydropyridyl)-glycine,        3-(4-tetrahydropyridyl)-alanine, N,N-gamma,        gamma′-diethyl-homoarginine. Included also are compounds such as        alpha-methyl-arginine, alpha-methyl-2,3-diaminopropionic acid,        alpha-methyl-histidine, alpha-methyl-ornithine where the alkyl        group occupies the pro-R position of the alpha-carbon. Also        included are the amides formed from alkyl, aromatic,        heteroaromatic (where the heteroaromatic group has one or more        nitrogens, oxygens or sulfur atoms singly or in combination)        carboxylic acids or any of the many well-known activated        derivatives such as acid chlorides, active esters, active        azolides and related derivatives) and lysine, ornithine, or        2,3-diaminopropionic acid;    -   d) substitution of acidic amino acids: including aspartic acid,        glutamic acid, homoglutamic acid, tyrosine, alkyl, aryl,        arylalkyl, and heteroaryl sulfonamides of 2,4-diaminopriopionic        acid, ornithine or lysine and tetrazole-substituted alkyl amino        acids;    -   e) substitution of side chain amide residues: including        asparagine, glutamine, and alkyl or aromatic substituted        derivatives of asparagine or glutamine; and    -   f) substitution of hydroxyl containing amino acids: including        serine, threonine, homoserine, 2,3-diaminopropionic acid, and        alkyl or aromatic substituted derivatives of serine or        threonine.

In some cases, a p75^(NTR)-derived peptide or a Rab31-derived peptidecomprises one or more naturally occurring non-genetically encodedL-amino acids, synthetic L-amino acids or D-enantiomers of an aminoacid. For example, a p75^(NTR)-derived peptide or a Rab31-derivedpeptide can comprise only D-amino acids. For example, ap75^(NTR)-derived peptide or a Rab31-derived peptide can comprise one ormore of the following residues: hydroxyproline, β-alanine,o-aminobenzoic acid, m-aminobenzoic acid, p-aminobenzoic acid,m-aminomethylbenzoic acid, 2,3-diaminopropionic acid, α-aminoisobutyricacid, N-methylglycine (sarcosine), ornithine, citrulline,t-butylalanine, t-butylglycine, N-methylisoleucine, phenylglycine,cyclohexylalanine, norleucine, naphthylalanine, pyridylalanine3-benzothienyl alanine, 4-chlorophenylalanine, 2-fluorophenylalanine,3-fluorophenylalanine, 4-fluorophenylalanine, penicillamine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, β-2-thienylalanine,methionine sulfoxide, homoarginine, N-acetyl lysine, 2,4-diamino butyricacid, rho-aminophenylalanine, N-methylvaline, homocysteine, homoserine,ε-amino hexanoic acid, ω-aminohexanoic acid, ω-aminoheptanoic acid,ω-aminooctanoic acid, ω-aminodecanoic acid, ω-aminotetradecanoic acid,cyclohexylalanine, α,γ-diaminobutyric acid, α,β-diaminopropionic acid,δ-amino valeric acid, and 2,3-diaminobutyric acid.

Other Modifications

A cysteine residue or a cysteine analog can be introduced into ap75^(NTR)-derived peptide or a Rab31-derived peptide to provide forlinkage to another peptide via a disulfide linkage or to provide forcyclization of the p75^(NTR)-derived peptide or Rab31-derived peptide.Methods of introducing a cysteine or cysteine analog are known in theart; see, e.g., U.S. Pat. No. 8,067,532.

A p75^(NTR)-derived peptide or a Rab31-derived peptide can be cyclized.One or more cysteine or cysteine analogs can be introduced into ap75^(NTR)-derived peptide or a Rab31-derived peptide, where theintroduced cysteine or cysteine analog can form a disulfide bond with asecond introduced cysteine or cysteine analog. Other means ofcyclization include introduction of an oxime linker or a lanthioninelinker; see, e.g., U.S. Pat. No. 8,044,175. Any combination of aminoacids (or non-amino acid moiety) that can form a cyclizing bond can beused and/or introduced. A cyclizing bond can be generated with anycombination of amino acids (or with amino acid and —(CH₂)_(n)—CO— or—(CH₂)_(n)—C₆H₄—CO—) with functional groups which allow for theintroduction of a bridge. Some examples are disulfides, disulfidemimetics such as the —(CH₂)_(n)— carba bridge, thioacetal, thioetherbridges (cystathionine or lanthionine) and bridges containing esters andethers.

Other modifications include, for example, an N-alkyl (or aryl)substitution (ψ[CONR]), or backbone crosslinking to construct lactamsand other cyclic structures. Other suitable derivatives of a peptideagent of the present disclosure include C-terminal hydroxymethylderivatives, O-modified derivatives (e.g., C-terminal hydroxymethylbenzyl ether), N-terminally modified derivatives including substitutedamides such as alkylamides and hydrazides.

In some cases, one or more L-amino acids in a p75^(NTR)-derived peptideor a Rab31-derived peptide is replaced with a D-amino acid.

In some cases, a p75^(NTR)-derived peptide or a Rab31-derived peptide isa retroinverso analog. Sela and Zisman (1997) FASEB J. 11:449.Retro-inverso peptide analogs are isomers of linear peptides in whichthe direction of the amino acid sequence is reversed (retro) and thechirality, D- or L-, of one or more amino acids therein is inverted(inverso) e.g., using D-amino acids rather than L-amino acids. See,e.g., Jameson et al. (1994) Nature 368:744; and Brady et al. (1994)Nature 368:692.

A p75^(NTR)-derived peptide or a Rab31-derived peptide can include aprotein transduction domain. “Protein Transduction Domain” or PTD refersto a polypeptide, polynucleotide, carbohydrate, or organic or inorganiccompound that facilitates traversing a lipid bilayer, micelle, cellmembrane, organelle membrane, or vesicle membrane. A PTD attached toanother molecule facilitates the molecule traversing a membrane, forexample going from extracellular space to intracellular space, orcytosol to within an organelle. In some embodiments, a PTD is covalentlylinked to the amino terminus of a p75^(NTR)-derived peptide or aRab31-derived peptide. In some embodiments, a PTD is covalently linkedto the carboxyl terminus of a p75^(NTR)-derived peptide or aRab31-derived peptide. Exemplary protein transduction domains includebut are not limited to a minimal undecapeptide protein transductiondomain (corresponding to residues 47-57 of HIV-1 TAT comprisingYGRKKRRQRRR (SEQ ID NO:36); a polyarginine sequence comprising a numberof arginines sufficient to direct entry into a cell (e.g., 3, 4, 5, 6,7, 8, 9, 10, or 10-50 arginines); a VP22 domain (Zender et al. (2002)Cancer Gene Ther. 9(6):489-96); an Drosophila Antennapedia proteintransduction domain (Noguchi et al. (2003) Diabetes 52(7):1732-1737); atruncated human calcitonin peptide (Trehin et al. (2004) Pharm. Research21:1248-1256); polylysine (Wender et al. (2000) Proc. Natl. Acad. Sci.USA 97:13003-13008); RRQRRTSKLMKR (SEQ ID NO:37); TransportanGWTLNSAGYLLGKINLKALAALAKKIL (SEQ ID NO:38);KALAWEAKLAKALAKALAKHLAKALAKALKCEA (SEQ ID NO:39); and RQIKIWFQNRRMKWKK(SEQ ID NO:40). Exemplary PTDs include but are not limited to,YGRKKRRQRRR (SEQ ID NO:36), RKKRRQRRR (SEQ ID NO:41); an argininehomopolymer of from 3 arginine residues to 50 arginine residues;Exemplary PTD domain amino acid sequences include, but are not limitedto, any of the following:

YGRKKRRQRRR; (SEQ ID NO: 36) RKKRRQRRR; (SEQ ID NO: 41) YARAAARQARA;(SEQ ID NO: 42) THRLPRRRRRR; (SEQ ID NO: 43) and GGRRARRRRRR.(SEQ ID NO: 44)

A subject p75NTR-derived peptide or a subject Rab31-derived peptide canbe modified by the covalent attachment of a water-soluble polymer suchas poly(ethylene glycol) (PEG), copolymers of PEG and polypropyleneglycol, polyvinylpyrrolidone or polyproline, carboxymethyl cellulose,dextran, polyvinyl alcohol, and the like. Such modifications also mayincrease the compound's solubility in aqueous solution. Polymers such asPEG may be covalently attached to one or more reactive amino residues,sulfhydryl residues or carboxyl residues. Numerous activated forms ofPEG have been described, including active esters of carboxylic acid orcarbonate derivatives, particularly those in which the leaving groupsare N-hydroxsuccinimide, p-nitrophenol, imdazole or1-hydroxy-2-nitrobenzene-3 sulfone for reaction with amino groups,multimode or halo acetyl derivatives for reaction with sulfhydrylgroups, and amino hydrazine or hydrazide derivatives for reaction withcarbohydrate groups.

Methods of Regulating Glucose Metabolism

The present disclosure provides methods of regulating glucosemetabolism. A subject method generally involves administering to anindividual an effective amount of an agent that reduces the level ofp75^(NTR) and/or reduces binding of p75^(NTR) to a Rab5 or a Rab31GTPase. A subject method can provide for one or more of: 1) increasingGDR; 2) increasing IS-GDR; 3) increasing insulin-stimulated glucoseuptake into adipocytes and myocytes; 4) reducing blood glucose level;and 5) increasing insulin sensitivity. Suitable agents include a subjectp75^(NTR)-derived peptide (as described above); a subject Rab31-derivedpeptide (as described above); and an interfering nucleic acid thatreduces the level of p75^(NTR) in a cell (as described below).

In some cases, an “effective amount” of an agent that reduces the levelof p75^(NTR) and/or reduces binding of p75^(NTR) to a Rab5 or a Rab31GTPase reduces a blood glucose level in an individual in need thereof byat least about 10%, at least about 15%, at least about 20%, at leastabout 25%, at least about 30%, at least about 35%, at least about 40%,at least about 45%, at least about 50%, at least about 55%, at leastabout 60%, at least about 65%, at least about 70%, or at least about75%, compared to the blood glucose level in the individual in theabsence of treatment with the agent.

In some cases, an “effective amount” of an agent that reduces the levelof p75^(NTR) and/or reduces binding of p75^(NTR) to a Rab5 or a Rab31GTPase increases the GDR or the IS-GDR in an individual in need thereofby at least about 10%, at least about 25%, at least about 50%, at leastabout 75%, at least about 2-fold, at least about 5-fold, or more than5-fold, compared to the GDR or the IS-GDR in the individual in theabsence of treatment with the agent.

In some cases, an “effective amount” of an agent that reduces the levelof p75^(NTR) and/or reduces binding of p75^(NTR) to a Rab5 or a Rab31GTPase increases insulin sensitivity an individual in need thereof by atleast about 10%, at least about 25%, at least about 50%, at least about75%, at least about 2-fold, at least about 5-fold, or more than 5-fold,compared to the insulin sensitivity in the individual in the absence oftreatment with the agent.

Interfering RNA

An agent that reduces the level of p75^(NTR) includes an interferingnucleic acid specific for p75^(NTR) (including a nucleic acid comprisinga nucleotide sequence encoding an interfering nucleic acid specific forp75^(NTR)).

An interfering nucleic acid specific for p75^(NTR) can reduce the levelof p75^(NTR) in a cell (e.g., an adipocyte; a myocyte) by at least about10%, at least about 15%, at least about 20%, at least about 25%, atleast about 30%, at least about 35%, at least about 40%, at least about45%, at least about 50%, at least about 55%, at least about 60%, atleast about 65%, at least about 70%, or at least about 75%, compared tothe level of p75^(NTR) in the cell in the absence of the interferingnucleic acid.

An interfering nucleic acid specific for p75^(NTR) can reduces a bloodglucose level in an individual in need thereof by at least about 10%, atleast about 15%, at least about 20%, at least about 25%, at least about30%, at least about 35%, at least about 40%, at least about 45%, atleast about 50%, at least about 55%, at least about 60%, at least about65%, at least about 70%, or at least about 75%, compared to the bloodglucose level in the individual not treated with the interfering nucleicacid.

In some cases, an interfering nucleic acid specific for p75^(NTR) is anshRNA, or a nucleic acid comprising a nucleotide sequence encoding anshRNA specific for p75^(NTR). In some cases a nucleotide sequenceencoding an shRNA specific for p75^(NTR) is operably linked to a controlelement (e.g., a promoter and/or an enhancer) that provides forselective expression in an adipocyte. In some cases a nucleotidesequence encoding an shRNA specific for p75^(NTR) is operably linked toa control element (e.g., a promoter and/or an enhancer) that providesfor selective expression in myocyte (muscle cell).

In some cases, a control element (e.g., a promoter and/or an enhancer)that provides for selective expression in a myocyte is a smooth musclecell-specific control element. Smooth muscle cell-specific controlelements include, e.g., a SM22α promoter (see, e.g., Akyürek et al.(2000) Mol. Med. 6:983; and U.S. Pat. No. 7,169,874); a smoothelinpromoter (see, e.g., WO 2001/018048); an α-smooth muscle actin promoter;etc. For example, a 0.4 kb region of the SM22α promoter, within whichlie two CArG elements, has been shown to mediate vascular smooth musclecell-specific expression (see, e.g., Kim, et al. (1997) Mol. Cell. Biol.17, 2266-2278; Li, et al., (1996) J. Cell Biol. 132, 849-859; andMoessler, et al. (1996) Development 122, 2415-2425).

In some cases, a promoter that provides for selective expression in amyocyte is a skeletal muscle cell-specific control element (e.g.,promoter and/or enhancer). Skeletal muscle cell-specific promotersinclude, but are not limited to, a Pitx3 promoter (Coulon et al. (2007)J. Biol. Chem. 282:33192); a Mef2c promoter (see, e.g., Heidt and Black(2005) Genesis 42:28); a desmin control element (see, e.g., Pacak et al.(2008) Genetic Vaccines and Therapy 6:13; and Talbot et al. (2010) Mol.Ther. 18:601); a pM or a pH control element from a human aldolase gene(see, e.g., Concordet et al. (1993) Mol. Cell. Biol. 13:9); cardiactroponin T gene promoter (see, e.g, Mar and Ordahl (1988) Proc. Natl.Acad. Sci. USA 85:6404); a muscle creatine kinase control element (see,e.g., Wang et al. (2008) Gene Ther. 15:1489); a skeletal α-actin controlelement (Ernst et al. (1991) Mol. Cell Biol. 11:3735); and the like.

Adipocyte-specific control elements can include, e.g., an aP2 genepromoter/enhancer, e.g., a region from −5.4 kb to +21 bp of a human aP2gene (see, e.g., Tozzo et al. (1997) Endocrinol. 138:1604; Ross et al.(1990) Proc. Natl. Acad. Sci. USA 87:9590; and Pavjani et al. (2005)Nat. Med. 11:797); a glucose transporter-4 (GLUT4) promoter (see, e.g.,Knight et al. (2003) Proc. Natl. Acad. Sci. USA 100:14725); a fatty acidtranslocase (FAT/CD36) promoter (see, e.g., Kuriki et al. (2002) Biol.Pharm. Bull. 25:1476; and Sato et al. (2002) J. Biol. Chem. 277:15703);a stearoyl-CoA desaturase-1 (SCD1) promoter (Tabor et al. (1999) J.Biol. Chem. 274:20603); a leptin promoter (see, e.g., Mason et al.(1998) Endocrinol. 139:1013; and Chen et al. (1999) Biochem. Biophys.Res. Comm. 262:187); an adiponectin promoter (see, e.g., Kita et al.(2005) Biochem. Biophys. Res. Comm. 331:484; and Chakrabarti (2010)Endocrinol. 151:2408); an adipsin promoter (see, e.g., Platt et al.(1989) Proc. Natl. Acad. Sci. USA 86:7490); a resistin promoter (see,e.g., Seo et al. (2003) Molec. Endocrinol. 17:1522); and the like.

Agents that reduce the level of p75^(NTR) in a cell (e.g., in anadipocyte; in a myocyte) include nucleic acid agents (“inhibitorynucleic acids”) that reduce the level of active of p75^(NTR) in a cell.Suitable agents that reduce the level of p75^(NTR) activity in a cell(e.g., that reduce the total amount of p75^(NTR) in a cell) includeinterfering nucleic acids, e.g., interfering RNA molecules. In oneembodiment, reduction of p75^(NTR) levels is accomplished through RNAinterference (RNAi) by contacting a cell with a small nucleic acidmolecule, such as a short interfering nucleic acid (siNA), a shortinterfering RNA (siRNA), a double-stranded RNA (dsRNA), a micro-RNA(miRNA), or a short hairpin RNA (shRNA) molecule, or modulation ofexpression of a small interfering RNA (siRNA) so as to provide fordecreased levels of p75^(NTR).

p75^(NTR)-specific interfering nucleic acids can be designed based onthe nucleotide sequence of a p75NTR-encoding nucleotide sequence. Forexample, in some embodiments, a p75NTR-encoding nucleotide sequence asset forth in FIG. 15 is used to design an interfering nucleic acid.

The term “short interfering nucleic acid,” “siNA,” “short interferingRNA,” “siRNA,” “short interfering nucleic acid molecule,” “shortinterfering oligonucleotide molecule,” or “chemically-modified shortinterfering nucleic acid molecule” as used herein refers to any nucleicacid molecule capable of inhibiting or down regulating gene expression,for example by mediating RNA interference “RNAi” or gene silencing in asequence-specific manner. Design of RNAi molecules when given a targetgene is routine in the art. See also US 2005/0282188 (which isincorporated herein by reference) as well as references cited therein.See, e.g., Pushparaj et al. Clin Exp Pharmacol Physiol. 2006 May-June;33(5-6):504-10; Lutzelberger et al. Handb Exp Pharmacol. 2006;(173):243-59; Aronin et al. Gene Ther. 2006 March; 13(6):509-16; Xie etal. Drug Discov Today. 2006 January; 11(1-2):67-73; Grunweller et al.Curr Med. Chem. 2005; 12(26):3143-61; and Pekaraik et al. Brain ResBull. 2005 Dec. 15; 68(1-2):115-20.

Methods for design and production of siRNAs to a desired target areknown in the art, and their application to p75NTR genes for the purposesdisclosed herein will be readily apparent to the ordinarily skilledartisan, as are methods of production of p75NTR having modifications(e.g., chemical modifications) to provide for, e.g., enhanced stability,bioavailability, and other properties to enhance use as therapeutics. Inaddition, methods for formulation and delivery of siRNAs to a subjectare also well known in the art. See, e.g., US 2005/0282188; US2005/0239731; US 2005/0234232; US 2005/0176018; US 2005/0059817; US2005/0020525; US 2004/0192626; US 2003/0073640; US 2002/0150936; US2002/0142980; and US2002/0120129, each of which is incorporated hereinby reference.

Publicly available tools to facilitate design of siRNAs are available inthe art. See, e.g., DEQOR: Design and Quality Control of RNAi (availableon the internet at cluster-1.mpi-cbg.de/Deqor/deqor.html). See also,Henschel et al. Nucleic Acids Res. 2004 Jul. 1; 32(Web Serverissue):W113-20. DEQOR is a web-based program which uses a scoring systembased on state-of-the-art parameters for siRNA design to evaluate theinhibitory potency of siRNAs. DEQOR, therefore, can help to predict (i)regions in a gene that show high silencing capacity based on the basepair composition and (ii) siRNAs with high silencing potential forchemical synthesis. In addition, each siRNA arising from the input queryis evaluated for possible cross-silencing activities by performing BLASTsearches against the transcriptome or genome of a selected organism.DEQOR can therefore predict the probability that an mRNA fragment willcross-react with other genes in the cell and helps researchers to designexperiments to test the specificity of siRNAs or chemically designedsiRNAs.

A suitable siRNA or shRNA sequence for reducing p75^(NTR) expressionincludes, e.g., 5′-GGAGACAUGUUCCACAGGCAUCGAAAUGCCUGUGGAACAUGUCUCCUU-3′(shRNA) (SEQ ID NO:56).

siNA molecules can be of any of a variety of forms. For example the siNAcan be a double-stranded polynucleotide molecule comprisingself-complementary sense and antisense regions, wherein the antisenseregion comprises nucleotide sequence that is complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof and thesense region having nucleotide sequence corresponding to the targetnucleic acid sequence or a portion thereof. siNA can also be assembledfrom two separate oligonucleotides, where one strand is the sense strandand the other is the antisense strand, wherein the antisense and sensestrands are self-complementary. In this embodiment, each strandgenerally comprises nucleotide sequence that is complementary tonucleotide sequence in the other strand; such as where the antisensestrand and sense strand form a duplex or double stranded structure, forexample wherein the double stranded region is about 15 base pairs toabout 30 base pairs, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprisesnucleotide sequence that is complementary to nucleotide sequence in atarget nucleic acid molecule or a portion thereof and the sense strandcomprises nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof (e.g., about 15 nucleotides to about 25 ormore nucleotides of the siNA molecule are complementary to the targetnucleic acid or a portion thereof).

Alternatively, the siNA can be assembled from a single oligonucleotide,where the self-complementary sense and antisense regions of the siNA arelinked by a nucleic acid-based or non-nucleic acid-based linker(s). ThesiNA can be a polynucleotide with a duplex, asymmetric duplex, hairpinor asymmetric hairpin secondary structure, having self-complementarysense and antisense regions, wherein the antisense region comprisesnucleotide sequence that is complementary to nucleotide sequence in aseparate target nucleic acid molecule or a portion thereof and the senseregion having nucleotide sequence corresponding to the target nucleicacid sequence or a portion thereof.

The siNA can be a circular single-stranded polynucleotide having two ormore loop structures and a stem comprising self-complementary sense andantisense regions, wherein the antisense region comprises nucleotidesequence that is complementary to nucleotide sequence in a targetnucleic acid molecule or a portion thereof and the sense region havingnucleotide sequence corresponding to the target nucleic acid sequence ora portion thereof, and wherein the circular polynucleotide can beprocessed either in vivo or in vitro to generate an active siNA moleculecapable of mediating RNAi. The siNA can also comprise a single strandedpolynucleotide having nucleotide sequence complementary to nucleotidesequence in a target nucleic acid molecule or a portion thereof (e.g.,where such siNA molecule does not require the presence within the siNAmolecule of nucleotide sequence corresponding to the target nucleic acidsequence or a portion thereof), wherein the single strandedpolynucleotide can further comprise a terminal phosphate group, such asa 5′-phosphate (see for example Martinez et al., 2002, Cell., 110,563-574 and Schwarz et al., 2002, Molecular Cell, 10, 537-568), or5′,3′-diphosphate.

In certain embodiments, the siNA molecule contains separate sense andantisense sequences or regions, wherein the sense and antisense regionsare covalently linked by nucleotide or non-nucleotide linkers moleculesas is known in the art, or are alternately non-covalently linked byionic interactions, hydrogen bonding, van der Waals interactions,hydrophobic interactions, and/or stacking interactions. In certainembodiments, the siNA molecules comprise nucleotide sequence that iscomplementary to nucleotide sequence of a target gene. In anotherembodiment, the siNA molecule interacts with nucleotide sequence of atarget gene in a manner that causes inhibition of expression of thetarget gene.

As used herein, siNA molecules need not be limited to those moleculescontaining only RNA, but further encompasses chemically-modifiednucleotides and non-nucleotides. In certain embodiments, the shortinterfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containingnucleotides. siNAs do not necessarily require the presence ofnucleotides having a 2′-hydroxy group for mediating RNAi and as such,siNA molecules optionally do not include any ribonucleotides (e.g.,nucleotides having a 2′-OH group). Such siNA molecules that do notrequire the presence of ribonucleotides within the siNA molecule tosupport RNAi can however have an attached linker or linkers or otherattached or associated groups, moieties, or chains containing one ormore nucleotides with 2′-OH groups. Optionally, siNA molecules cancomprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of thenucleotide positions. The modified short interfering nucleic acidmolecules can also be referred to as short interfering modifiedoligonucleotides “siMON.”

As used herein, the term siNA is meant to be equivalent to other termsused to describe nucleic acid molecules that are capable of mediatingsequence specific RNAi, for example short interfering RNA (siRNA),double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA(shRNA), short interfering oligonucleotide, short interfering nucleicacid, short interfering modified oligonucleotide, chemically-modifiedsiRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. Inaddition, as used herein, the term RNAi is meant to be equivalent toother terms used to describe sequence specific RNA interference, such aspost transcriptional gene silencing, translational inhibition, orepigenetics. For example, siNA molecules can be used to epigeneticallysilence a target gene at the post-transcriptional level, thepre-transcriptional level, or both the post-transcriptional andpre-transcriptional levels. In a non-limiting example, epigeneticregulation of gene expression by siNA molecules can result from siNAmediated modification of chromatin structure or methylation pattern toalter gene expression (see, for example, Verdel et al., 2004, Science,303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire,2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297,1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al.,2002, Science, 297, 2232-2237).

siNA molecules contemplated herein can comprise a duplex formingoligonucleotide (DFO) see, e.g., WO 05/019453; and US 2005/0233329,which are incorporated herein by reference). siNA molecules alsocontemplated herein include multifunctional siNA, (see, e.g., WO05/019453 and US 2004/0249178). The multifunctional siNA can comprisesequence targeting, for example, two regions of p75NTR.

siNA molecules contemplated herein can comprise an asymmetric hairpin orasymmetric duplex. By “asymmetric hairpin” as used herein is meant alinear siNA molecule comprising an antisense region, a loop portion thatcan comprise nucleotides or non-nucleotides, and a sense region thatcomprises fewer nucleotides than the antisense region to the extent thatthe sense region has enough complementary nucleotides to base pair withthe antisense region and form a duplex with loop. For example, anasymmetric hairpin siNA molecule can comprise an antisense region havinglength sufficient to mediate RNAi in a cell or in vitro system (e.g.about 15 to about 30, or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24,25, 26, 27, 28, 29, or 30 nucleotides) and a loop region comprisingabout 4 to about 12 (e.g., about 4, 5, 6, 7, 8, 9, 10, 11, or 12)nucleotides, and a sense region having about 3 to about 25 (e.g., about3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, or 25) nucleotides that are complementary to the antisenseregion. The asymmetric hairpin siNA molecule can also comprise a5′-terminal phosphate group that can be chemically modified. The loopportion of the asymmetric hairpin siNA molecule can comprisenucleotides, non-nucleotides, linker molecules, or conjugate moleculesas described herein.

By “asymmetric duplex” as used herein is meant a siNA molecule havingtwo separate strands comprising a sense region and an antisense region,wherein the sense region comprises fewer nucleotides than the antisenseregion to the extent that the sense region has enough complementarynucleotides to base pair with the antisense region and form a duplex.For example, an asymmetric duplex siNA molecule can comprise anantisense region having length sufficient to mediate RNAi in a cell orin vitro system (e.g. about 15 to about 30, or about 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or nucleotides) and a senseregion having about 3 to about 25 (e.g., about 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25)nucleotides that are complementary to the antisense region.

Stability and/or half-life of siRNAs can be improved through chemicallysynthesizing nucleic acid molecules with modifications (base, sugarand/or phosphate) can prevent their degradation by serum ribonucleases,which can increase their potency (see e.g., Eckstein et al.,International Publication No. WO 92/07065; Perrault et al., 1990 Nature344, 565; Pieken et al., 1991, Science 253, 314; Usman and Cedergren,1992, Trends in Biochem. Sci. 17, 334; Usman et al., InternationalPublication No. WO 93/15187; and Rossi et al., International PublicationNo. WO 91/03162; Sproat, U.S. Pat. No. 5,334,711; Gold et al., U.S. Pat.No. 6,300,074; and Burgin et al., supra; all of which are incorporatedby reference herein, describing various chemical modifications that canbe made to the base, phosphate and/or sugar moieties of the nucleic acidmolecules described herein. Modifications that enhance their efficacy incells, and removal of bases from nucleic acid molecules to shortenoligonucleotide synthesis times and reduce chemical requirements aredesired.

For example, oligonucleotides are modified to enhance stability and/orenhance biological activity by modification with nuclease resistantgroups, for example, 2′-amino, 2′-C-allyl, 2′-fluoro, 2′-O-methyl,2′-O-allyl, 2′-H, nucleotide base modifications (for a review see Usmanand Cedergren, 1992, TIBS. 17, 34; Usman et al., 1994, Nucleic AcidsSymp. Ser. 31, 163; Burgin et al., 1996, Biochemistry, 35, 14090). Sugarmodification of nucleic acid molecules have been extensively describedin the art (see Eckstein et al., International Publication PCT No. WO92/07065; Perrault et al. Nature, 1990, 344, 565-568; Pieken et al.Science, 1991, 253, 314-317; Usman and Cedergren, Trends in Biochem.Sci., 1992, 17, 334-339; Usman et al. International Publication PCT No.WO 93/15187; Sproat, U.S. Pat. No. 5,334,711 and Beigelman et al., 1995,J. Biol. Chem., 270, 25702; Beigelman et al., International PCTpublication No. WO 97/26270; Beigelman et al., U.S. Pat. No. 5,716,824;Usman et al., U.S. Pat. No. 5,627,053; Woolf et al., International PCTPublication No. WO 98/13526; Thompson et al., U.S. Ser. No. 60/082,404which was filed on Apr. 20, 1998; Karpeisky et al., 1998, TetrahedronLett., 39, 1131; Eamshaw and Gait, 1998, Biopolymers (Nucleic AcidSciences), 48, 39-55; Verma and Eckstein, 1998, Annu Rev. Biochem., 67,99-134; and Burlina et al., 1997, Bioorg. Med. Chem., 5, 1999-2010; eachof which are hereby incorporated in their totality by reference herein).In view of such teachings, similar modifications can be used asdescribed herein to modify the siNA nucleic acid molecules of disclosedherein so long as the ability of siNA to promote RNAi is cells is notsignificantly inhibited.

Short interfering nucleic acid (siNA) molecules having chemicalmodifications that maintain or enhance activity are contemplated herein.Such a nucleic acid is also generally more resistant to nucleases thanan unmodified nucleic acid. Accordingly, the in vitro and/or in vivoactivity should not be significantly lowered. Nucleic acid moleculesdelivered exogenously are generally selected to be stable within cellsat least for a period sufficient for transcription and/or translation ofthe target RNA to occur and to provide for modulation of production ofthe encoded mRNA and/or polypeptide so as to facilitate reduction of thelevel of the target gene product.

Production of RNA and DNA molecules can be accomplished syntheticallyand can provide for introduction of nucleotide modifications to providefor enhanced nuclease stability. (see, e.g., Wincott et al., 1995,Nucleic Acids Res. 23, 2677; Caruthers et al., 1992, Methods inEnzymology 211, 3-19, incorporated by reference herein. In oneembodiment, nucleic acid molecules include one or more (e.g., about 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more) G-clamp nucleotides, which aremodified cytosine analogs which confer the ability to hydrogen bond bothWatson-Crick and Hoogsteen faces of a complementary guanine within aduplex, and can provide for enhanced affinity and specificity to nucleicacid targets (see, e.g., Lin et al. 1998, J. Am. Chem. Soc., 120,8531-8532). In another example, nucleic acid molecules can include oneor more (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) LNA “lockednucleic acid” nucleotides such as a 2′,4′-C methylene bicyclo nucleotide(see, e.g., Wengel et al., WO 00/66604 and WO 99/14226).

siNA molecules can be provided as conjugates and/or complexes, e.g., tofacilitate delivery of siNA molecules into a cell. Exemplary conjugatesand/or complexes include those composed of an siNA and a small molecule,lipid, cholesterol, phospholipid, nucleoside, antibody, toxin,negatively charged polymer (e.g., protein, peptide, hormone,carbohydrate, polyethylene glycol, or polyamine). In general, thetransporters described are designed to be used either individually or aspart of a multi-component system, with or without degradable linkers.These compounds can improve delivery and/or localization of nucleic acidmolecules into cells in the presence or absence of serum (see, e.g.,U.S. Pat. No. 5,854,038). Conjugates of the molecules described hereincan be attached to biologically active molecules via linkers that arebiodegradable, such as biodegradable nucleic acid linker molecules.

Formulations, Dosages, and Routes of Administration

A p75^(NTR)-derived peptide, or a Rab31-derived peptide, or ap75^(NTR)-specific interfering RNA can be formulated with one or morepharmaceutically acceptable excipients. p75^(NTR)-derived peptides,Rab31-derived peptides, and p75^(NTR)-specific interfering RNAs arereferred to generically below as “active agent” or “drug.” Thus, thepresent disclosure provides a pharmaceutical composition comprising: a)a subject p75^(NTR)-derived peptide; and b) a pharmaceuticallyacceptable excipient. The present disclosure further provides apharmaceutical composition comprising: a) a p75^(NTR)-specificinterfering RNA (including a nucleic acid comprising a nucleotidesequence encoding a p75^(NTR)-specific interfering RNA); and b) apharmaceutically acceptable excipient. The present disclosure furtherprovides a pharmaceutical composition comprising: a) a Rab31-derivedpeptide; and b) a pharmaceutically acceptable excipient.

A wide variety of pharmaceutically acceptable excipients are known inthe art and need not be discussed in detail herein. Pharmaceuticallyacceptable excipients have been amply described in a variety ofpublications, including, for example, A. Gennaro (2000) “Remington: TheScience and Practice of Pharmacy,” 20th edition, Lippincott, Williams, &Wilkins; Pharmaceutical Dosage Forms and Drug Delivery Systems (1999) H.C. Ansel et al., eds., 7th ed., Lippincott, Williams, & Wilkins; andHandbook of Pharmaceutical Excipients (2000) A. H. Kibbe et al., eds.,3rd ed. Amer. Pharmaceutical Assoc.

The pharmaceutically acceptable excipients, such as vehicles, adjuvants,carriers or diluents, are readily available to the public. Moreover,pharmaceutically acceptable auxiliary substances, such as pH adjustingand buffering agents, tonicity adjusting agents, stabilizers, wettingagents and the like, are readily available to the public.

The term “unit dosage form,” as used herein, refers to physicallydiscrete units suitable as unitary dosages for human and animalsubjects, each unit containing a predetermined quantity of a subjectactive agent calculated in an amount sufficient to produce the desiredeffect in association with a pharmaceutically acceptable diluent,carrier or vehicle. The specifications for an active agent depend on theparticular compound employed and the effect to be achieved, and thepharmacodynamics associated with each compound in the host.

In the subject methods, a p75^(NTR)-derived peptide, or a Rab31-derivedpeptide, or a p75^(NTR)-specific interfering RNA may be administered tothe host using any convenient means capable of resulting in the desiredoutcome, e.g., reduction of disease, reduction of a symptom of adisease, reduction of blood glucose levels, etc. Thus, ap75^(NTR)-derived peptide, a Rab31-derived peptide, or ap75^(NTR)-specific interfering RNA can be incorporated into a variety offormulations for therapeutic administration. More particularly, ap75^(NTR)-derived peptide, a Rab31-derived peptide, or ap75^(NTR)-specific interfering RNA can be formulated into pharmaceuticalcompositions by combination with appropriate, pharmaceuticallyacceptable carriers or diluents, and may be formulated into preparationsin solid, semi-solid, liquid or gaseous forms, such as tablets,capsules, powders, granules, ointments, solutions, suppositories,injections, inhalants and aerosols.

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of the agent adequate to achieve thedesired state in the subject being treated.

In pharmaceutical dosage forms, a p75^(NTR)-derived peptide, aRab31-derived peptide, or a p75^(NTR)-specific interfering RNA (“activeagent”) may be administered in the form of its pharmaceuticallyacceptable salts, or an active agent may be used alone or in appropriateassociation, as well as in combination, with other pharmaceuticallyactive compounds. The following methods and excipients are merelyexemplary and are in no way limiting.

For oral preparations, an active agent (a p75^(NTR)-derived peptide, aRab31-derived peptide, or a p75^(NTR)-specific interfering RNA) can beused alone or in combination with appropriate additives to make tablets,powders, granules or capsules, for example, with conventional additives,such as lactose, mannitol, corn starch or potato starch; with binders,such as crystalline cellulose, cellulose derivatives, acacia, cornstarch or gelatins; with disintegrators, such as corn starch, potatostarch or sodium carboxymethylcellulose; with lubricants, such as talcor magnesium stearate; and if desired, with diluents, buffering agents,moistening agents, preservatives and flavoring agents.

An active agent can be formulated into preparations for injection bydissolving, suspending or emulsifying them in an aqueous or nonaqueoussolvent, such as vegetable or other similar oils, synthetic aliphaticacid glycerides, esters of higher aliphatic acids or propylene glycol;and if desired, with conventional additives such as solubilizers,isotonic agents, suspending agents, emulsifying agents, stabilizers andpreservatives.

An active agent can be utilized in aerosol formulation to beadministered via inhalation. An active agent can be formulated intopressurized acceptable propellants such as dichlorodifluoromethane,propane, nitrogen and the like.

Furthermore, an active agent can be made into suppositories by mixingwith a variety of bases such as emulsifying bases or water-solublebases. An active agent can be administered rectally via a suppository.The suppository can include vehicles such as cocoa butter, carbowaxesand polyethylene glycol monomethyl ethers, which melt at bodytemperature, yet are solidified at room temperature.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the subject active agent. Similarly, unit dosageforms for injection or intravenous administration may comprise a subjectactive agent in a composition as a solution in sterile water, normalsaline or another pharmaceutically acceptable carrier.

An active agent can be formulated for administration by injection.Typically, injectable compositions are prepared as liquid solutions orsuspensions; solid forms suitable for solution in, or suspension in,liquid vehicles prior to injection may also be prepared. The preparationmay also be emulsified or the active ingredient encapsulated in liposomevehicles.

In some embodiments, an active agent is delivered by a continuousdelivery system. The term “continuous delivery system” is usedinterchangeably herein with “controlled delivery system” and encompassescontinuous (e.g., controlled) delivery devices (e.g., pumps) incombination with catheters, injection devices, and the like, a widevariety of which are known in the art.

Mechanical or electromechanical infusion pumps can also be suitable foruse. Examples of such devices include those described in, for example,U.S. Pat. Nos. 4,692,147; 4,360,019; 4,487,603; 4,360,019; 4,725,852;5,820,589; 5,643,207; 6,198,966; and the like. In general, delivery ofactive agent can be accomplished using any of a variety of refillable,pump systems. Pumps provide consistent, controlled release over time. Insome embodiments, the active agent is in a liquid formulation in adrug-impermeable reservoir, and is delivered in a continuous fashion tothe individual.

In one embodiment, the drug delivery system is an at least partiallyimplantable device. The implantable device can be implanted at anysuitable implantation site using methods and devices well known in theart. An implantation site is a site within the body of a subject atwhich a drug delivery device is introduced and positioned. Implantationsites include, but are not necessarily limited to a subdermal,subcutaneous, intramuscular, or other suitable site within a subject'sbody. Subcutaneous implantation sites are used in some embodimentsbecause of convenience in implantation and removal of the drug deliverydevice.

Drug release devices suitable for use may be based on any of a varietyof modes of operation. For example, the drug release device can be basedupon a diffusive system, a convective system, or an erodible system(e.g., an erosion-based system). For example, the drug release devicecan be an electrochemical pump, osmotic pump, an electroosmotic pump, avapor pressure pump, or osmotic bursting matrix, e.g., where the drug(“active agent”) is incorporated into a polymer and the polymer providesfor release of drug formulation concomitant with degradation of adrug-impregnated polymeric material (e.g., a biodegradable,drug-impregnated polymeric material). In other embodiments, the drugrelease device is based upon an electrodiffusion system, an electrolyticpump, an effervescent pump, a piezoelectric pump, a hydrolytic system,etc.

Drug release devices based upon a mechanical or electromechanicalinfusion pump can also be suitable for use. Examples of such devicesinclude those described in, for example, U.S. Pat. Nos. 4,692,147;4,360,019; 4,487,603; 4,360,019; 4,725,852, and the like. In general, asubject treatment method can be accomplished using any of a variety ofrefillable, non-exchangeable pump systems. Pumps and other convectivesystems are generally preferred due to their generally more consistent,controlled release over time. Osmotic pumps are used in some embodimentsdue to their combined advantages of more consistent controlled releaseand relatively small size (see, e.g., PCT published application no. WO97/27840 and U.S. Pat. Nos. 5,985,305 and 5,728,396)). Exemplaryosmotically-driven devices suitable for use include, but are notnecessarily limited to, those described in U.S. Pat. Nos. 3,760,984;3,845,770; 3,916,899; 3,923,426; 3,987,790; 3,995,631; 3,916,899;4,016,880; 4,036,228; 4,111,202; 4,111,203; 4,203,440; 4,203,442;4,210,139; 4,327,725; 4,627,850; 4,865,845; 5,057,318; 5,059,423;5,112,614; 5,137,727; 5,234,692; 5,234,693; 5,728,396; and the like.

In some embodiments, the drug delivery device is an implantable device.The drug delivery device can be implanted at any suitable implantationsite using methods and devices well known in the art. As noted infra, animplantation site is a site within the body of a subject at which a drugdelivery device is introduced and positioned. Implantation sitesinclude, but are not necessarily limited to a subdermal, subcutaneous,intramuscular, or other suitable site within a subject's body.

In some embodiments, an active agent (a p75^(NTR)-derived peptide, aRab31-derived peptide, or a p75^(NTR)-specific interfering RNA) isdelivered using an implantable drug delivery system, e.g., a system thatis programmable to provide for administration of an active agent.Exemplary programmable, implantable systems include implantable infusionpumps. Exemplary implantable infusion pumps, or devices useful inconnection with such pumps, are described in, for example, U.S. Pat.Nos. 4,350,155; 5,443,450; 5,814,019; 5,976,109; 6,017,328; 6,171,276;6,241,704; 6,464,687; 6,475,180; and 6,512,954. A further exemplarydevice that can be adapted for use is the Synchromed infusion pump(Medtronic).

Suitable excipient vehicles are, for example, water, saline, dextrose,glycerol, ethanol, or the like, and combinations thereof. In addition,if desired, the vehicle may contain minor amounts of auxiliarysubstances such as wetting or emulsifying agents or pH buffering agents.Actual methods of preparing such dosage forms are known, or will beapparent, to those skilled in the art. See, e.g., Remington'sPharmaceutical Sciences, Mack Publishing Company, Easton, Pa., 17thedition, 1985. The composition or formulation to be administered will,in any event, contain a quantity of an active agent adequate to achievethe desired state in the subject being treated.

Oral Formulations

In some embodiments, an active agent (a p75^(NTR)-derived peptide, aRab31-derived peptide, or a p75^(NTR)-specific interfering RNA) isformulated for oral delivery to an individual in need of such an agent.

For oral delivery, a formulation comprising an active agent will in someembodiments include an enteric-soluble coating material. Suitableenteric-soluble coating material include hydroxypropyl methylcelluloseacetate succinate (HPMCAS), hydroxypropyl methyl cellulose phthalate(HPMCP), cellulose acetate phthalate (CAP), polyvinyl phthalic acetate(PVPA), Eudragit™, and shellac.

As one non-limiting example of a suitable oral formulation, an activeagent is formulated with one or more pharmaceutical excipients andcoated with an enteric coating, as described in U.S. Pat. No. 6,346,269.For example, a solution comprising an active agent and a stabilizer iscoated onto a core comprising pharmaceutically acceptable excipients, toform an active agent-coated core; a sub-coating layer is applied to theactive agent-coated core, which is then coated with an enteric coatinglayer. The core generally includes pharmaceutically inactive componentssuch as lactose, a starch, mannitol, sodium carboxymethyl cellulose,sodium starch glycolate, sodium chloride, potassium chloride, pigments,salts of alginic acid, talc, titanium dioxide, stearic acid, stearate,micro-crystalline cellulose, glycerin, polyethylene glycol, triethylcitrate, tributyl citrate, propanyl triacetate, dibasic calciumphosphate, tribasic sodium phosphate, calcium sulfate, cyclodextrin, andcastor oil. Suitable solvents for an active agent include aqueoussolvents. Suitable stabilizers include alkali-metals and alkaline earthmetals, bases of phosphates and organic acid salts and organic amines.The sub-coating layer comprises one or more of an adhesive, aplasticizer, and an anti-tackiness agent. Suitable anti-tackiness agentsinclude talc, stearic acid, stearate, sodium stearyl fumarate, glycerylbehenate, kaolin and aerosil. Suitable adhesives include polyvinylpyrrolidone (PVP), gelatin, hydroxyethyl cellulose (HEC), hydroxypropylcellulose (HPC), hydroxypropyl methyl cellulose (HPMC), vinyl acetate(VA), polyvinyl alcohol (PVA), methyl cellulose (MC), ethyl cellulose(EC), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalates (CAP), xanthan gum, alginic acid, salts of alginicacid, Eudragit™, copolymer of methyl acrylic acid/methyl methacrylatewith polyvinyl acetate phthalate (PVAP). Suitable plasticizers includeglycerin, polyethylene glycol, triethyl citrate, tributyl citrate,propanyl triacetate and castor oil. Suitable enteric-soluble coatingmaterial include hydroxypropyl methylcellulose acetate succinate(HPMCAS), hydroxypropyl methyl cellulose phthalate (HPMCP), celluloseacetate phthalate (CAP), polyvinyl phthalic acetate (PVPA), Eudragit™and shellac.

Suitable oral formulations also include an active agent formulated withany of the following: microgranules (see, e.g., U.S. Pat. No.6,458,398); biodegradable macromers (see, e.g., U.S. Pat. No.6,703,037); biodegradable hydrogels (see, e.g., Graham and McNeill(1989) Biomaterials 5:27-36); biodegradable particulate vectors (see,e.g., U.S. Pat. No. 5,736,371); bioabsorbable lactone polymers (see,e.g., U.S. Pat. No. 5,631,015); slow release protein polymers (see,e.g., U.S. Pat. No. 6,699,504; Pelias Technologies, Inc.); apoly(lactide-co-glycolide/polyethylene glycol block copolymer (see,e.g., U.S. Pat. No. 6,630,155; Atrix Laboratories, Inc.); a compositioncomprising a biocompatible polymer and particles of metalcation-stabilized agent dispersed within the polymer (see, e.g., U.S.Pat. No. 6,379,701; Alkermes Controlled Therapeutics, Inc.); andmicrospheres (see, e.g., U.S. Pat. No. 6,303,148; Octoplus, B.V.).

Suitable oral formulations also include an active agent with any of thefollowing: a carrier such as Emisphere® (Emisphere Technologies, Inc.);TIMERx, a hydrophilic matrix combining xanthan and locust bean gumswhich, in the presence of dextrose, form a strong binder gel in water(Penwest); Geminex™ (Penwest); Procise™ (GlaxoSmithKline); SAVIT™(Mistral Pharma Inc.); RingCap™ (Alza Corp.); Smartrix® (SmartrixTechnologies, Inc.); SQZgel™ (MacroMed, Inc.); Geomatrix™ (Skye Pharma,Inc.); Oros® Tri-layer (Alza Corporation); and the like.

Also suitable for use are formulations such as those described in U.S.Pat. No. 6,296,842 (Alkermes Controlled Therapeutics, Inc.); U.S. Pat.No. 6,187,330 (Scios, Inc.); and the like.

Also suitable for use herein are formulations comprising an intestinalabsorption enhancing agent, and an active agent (e.g., ap75^(NTR)-derived peptide or a p75^(NTR)-specific interfering RNA).Suitable intestinal absorption enhancers include, but are not limitedto, calcium chelators (e.g., citrate, ethylenediamine tetracetic acid);surfactants (e.g., sodium dodecyl sulfate, bile salts,palmitoylcarnitine, and sodium salts of fatty acids); toxins (e.g.,zonula occludens toxin); and the like.

Inhalational Formulations

An active agent (a p75^(NTR)-derived peptide, a Rab31-derived peptide,or a p75^(NTR)-specific interfering RNA) will in some embodiments beadministered to a patient by means of a pharmaceutical delivery systemfor the inhalation route. An active agent can be formulated in a formsuitable for administration by inhalation. The inhalational route ofadministration provides the advantage that the inhaled drug can bypassthe blood-brain barrier. The pharmaceutical delivery system is one thatis suitable for respiratory therapy by delivery of an active agent tomucosal linings of the bronchi. A system that depends on the power of acompressed gas to expel the active agent from a container can be used.An aerosol or pressurized package can be employed for this purpose.

As used herein, the term “aerosol” is used in its conventional sense asreferring to very fine liquid or solid particles carries by a propellantgas under pressure to a site of therapeutic application. When apharmaceutical aerosol is employed, the aerosol contains an activeagent, which can be dissolved, suspended, or emulsified in a mixture ofa fluid carrier and a propellant. The aerosol can be in the form of asolution, suspension, emulsion, powder, or semi-solid preparation.Aerosols employed for use herein are intended for administration asfine, solid particles or as liquid mists via the respiratory tract of apatient. Various types of propellants known to one of skill in the artcan be utilized. Suitable propellants include, but are not limited to,hydrocarbons or other suitable gas. In the case of the pressurizedaerosol, the dosage unit may be determined by providing a value todeliver a metered amount.

An active agent can also be formulated for delivery with a nebulizer,which is an instrument that generates very fine liquid particles ofsubstantially uniform size in a gas. For example, a liquid containingthe active agent is dispersed as droplets. The small droplets can becarried by a current of air through an outlet tube of the nebulizer. Theresulting mist penetrates into the respiratory tract of the patient.

A powder composition containing an active agent, with or without alubricant, carrier, or propellant, can be administered to a mammal inneed of therapy. This embodiment of the present disclosure can becarried out with a conventional device for administering a powderpharmaceutical composition by inhalation. For example, a powder mixtureof the compound and a suitable powder base such as lactose or starch maybe presented in unit dosage form in for example capsular or cartridges,e.g. gelatin, or blister packs, from which the powder may beadministered with the aid of an inhaler.

There are several different types of inhalation methodologies which canbe employed in connection with the present disclosure. An active agentcan be formulated in basically three different types of formulations forinhalation. First, an active agent can be formulated with low boilingpoint propellants. Such formulations are generally administered byconventional meter dose inhalers (MDI's). However, conventional MDI'scan be modified so as to increase the ability to obtain repeatabledosing by utilizing technology which measures the inspiratory volume andflow rate of the patient as discussed within U.S. Pat. Nos. 5,404,871and 5,542,410.

Alternatively, an active agent can be formulated in aqueous or ethanolicsolutions and delivered by conventional nebulizers. In some embodiments,such solution formulations are aerosolized using devices and systemssuch as disclosed within U.S. Pat. Nos. 5,497,763; 5,544,646; 5,718,222;and 5,660,166.

An active agent can be formulated into dry powder formulations. Suchformulations can be administered by simply inhaling the dry powderformulation after creating an aerosol mist of the powder. Technology forcarrying such out is described within U.S. Pat. No. 5,775,320 and U.S.Pat. No. 5,740,794.

Dosages and Dosing

Depending on the subject and condition being treated and on theadministration route, an active agent (a p75^(NTR)-derived peptide, aRab31-derived peptide, or a p75^(NTR)-specific interfering RNA) can beadministered in dosages of, for example, 0.1 μg to 500 mg/kg body weightper day, e.g., from about 0.1 μg/kg body weight per day to about 1 μg/kgbody weight per day, from about 1 μg/kg body weight per day to about 25μg/kg body weight per day, from about 25 μg/kg body weight per day toabout 50 μg/kg body weight per day, from about 50 μg/kg body weight perday to about 100 μg/kg body weight per day, from about 100 μg/kg bodyweight per day to about 500 μg/kg body weight per day, from about 500μg/kg body weight per day to about 1 mg/kg body weight per day, fromabout 1 mg/kg body weight per day to about 25 mg/kg body weight per day,from about 25 mg/kg body weight per day to about 50 mg/kg body weightper day, from about 50 mg/kg body weight per day to about 100 mg/kg bodyweight per day, from about 100 mg/kg body weight per day to about 250mg/kg body weight per day, or from about 250 mg/kg body weight per dayto about 500 mg/kg body weight per day. The range is broad, since ingeneral the efficacy of a therapeutic effect for different mammalsvaries widely with doses typically being 20, 30 or even 40 times smaller(per unit body weight) in man than in the rat. Similarly the mode ofadministration can have a large effect on dosage. Thus, for example,oral dosages may be about ten times the injection dose. Higher doses maybe used for localized routes of delivery.

For example, an active agent can be administered in an amount of fromabout 1 mg to about 1000 mg per dose, e.g., from about 1 mg to about 5mg, from about 5 mg to about 10 mg, from about 10 mg to about 20 mg,from about 20 mg to about 25 mg, from about 25 mg to about 50 mg, fromabout 50 mg to about 75 mg, from about 75 mg to about 100 mg, from about100 mg to about 125 mg, from about 125 mg to about 150 mg, from about150 mg to about 175 mg, from about 175 mg to about 200 mg, from about200 mg to about 225 mg, from about 225 mg to about 250 mg, from about250 mg to about 300 mg, from about 300 mg to about 350 mg, from about350 mg to about 400 mg, from about 400 mg to about 450 mg, from about450 mg to about 500 mg, from about 500 mg to about 750 mg, or from about750 mg to about 1000 mg per dose.

An exemplary dosage may be a solution suitable for intravenousadministration; a tablet taken from two to six times daily, or onetime-release capsule or tablet taken once a day and containing aproportionally higher content of active agent, etc. The time-releaseeffect may be obtained by capsule materials that dissolve at differentpH values, by capsules that release slowly by osmotic pressure, or byany other known means of controlled release.

Those of skill in the art will readily appreciate that dose levels canvary as a function of the specific compound, the severity of thesymptoms and the susceptibility of the subject to side effects.Preferred dosages for a given compound are readily determinable by thoseof skill in the art by a variety of means.

Although the dosage used will vary depending on the clinical goals to beachieved, a suitable dosage range is in some embodiments one whichprovides up to about 1 μg to about 1,000 μg or about 10,000 μg of activeagent in a blood sample taken from the individual being treated, about24 hours after administration of the active agent to the individual.

Unit dosage forms for oral or rectal administration such as syrups,elixirs, and suspensions may be provided wherein each dosage unit, forexample, teaspoonful, tablespoonful, tablet or suppository, contains apredetermined amount of the composition containing one or more activeagents. Similarly, unit dosage forms for injection or intravenousadministration may comprise the active agent(s) in a composition as asolution in sterile water, normal saline or another pharmaceuticallyacceptable carrier.

In some embodiments, multiple doses of an active agent are administered.The frequency of administration of an active agent can vary depending onany of a variety of factors, e.g., severity of the symptoms, etc. Forexample, in some embodiments, an active agent is administered once permonth, twice per month, three times per month, every other week (qow),once per week (qw), twice per week (biw), three times per week (tiw),four times per week, five times per week, six times per week, everyother day (qod), daily (qd), twice a day (bid), or three times a day(tid). As discussed above, in some embodiments, an active agent isadministered continuously.

The duration of administration of an active agent, e.g., the period oftime over which an active agent is administered, can vary, depending onany of a variety of factors, e.g., patient response, etc. For example,an active agent can be administered over a period of time ranging fromabout one day to about one week, from about two weeks to about fourweeks, from about one month to about two months, from about two monthsto about four months, from about four months to about six months, fromabout six months to about eight months, from about eight months to about1 year, from about 1 year to about 2 years, or from about 2 years toabout 4 years, or more. In some embodiments, an active agent isadministered for the lifetime of the individual.

Routes of Administration

An active agent (a p75^(NTR)-derived peptide, a Rab31-derived peptide,or a p75^(NTR)-specific interfering RNA) is administered to anindividual using any available method and route suitable for drugdelivery, including in vivo and ex vivo methods, as well as systemic andlocalized routes of administration. Administration can be acute (e.g.,of short duration, e.g., a single administration, administration for oneday to one week), or chronic (e.g., of long duration, e.g.,administration for longer than one week, e.g., administration over aperiod of time of from about 2 weeks to about one month, from about onemonth to about 3 months, from about 3 months to about 6 months, fromabout 6 months to about 1 year, or longer than one year).

Conventional and pharmaceutically acceptable routes of administrationinclude intranasal, intramuscular, intratracheal, subcutaneous,intradermal, transdermal, sublingual, topical application, intravenous,ocular (e.g., topically to the eye, intravitreal, etc.), rectal, nasal,oral, and other enteral and parenteral routes of administration. Routesof administration may be combined, if desired, or adjusted dependingupon the agent and/or the desired effect. The active agent can beadministered in a single dose or in multiple doses.

An active agent can be administered to a host using any availableconventional methods and routes suitable for delivery of conventionaldrugs, including systemic or localized routes. In general, suitableroutes of administration include, but are not necessarily limited to,enteral, parenteral, and inhalational routes.

Parenteral routes of administration other than inhalation administrationinclude, but are not necessarily limited to, topical, transdermal,subcutaneous, intramuscular, intraorbital, intracapsular, intraspinal,intrasternal, ocular, and intravenous routes, i.e., any route ofadministration other than through the alimentary canal. Parenteraladministration can be carried to effect systemic or local delivery ofthe agent. Where systemic delivery is desired, administration typicallyinvolves invasive or systemically absorbed topical or mucosaladministration of pharmaceutical preparations.

An active agent can also be delivered to the subject by enteraladministration. Enteral routes of administration include, but are notnecessarily limited to, oral and rectal (e.g., using a suppository)delivery.

Methods of administration of an active agent through the skin or mucosainclude, but are not necessarily limited to, topical application of asuitable pharmaceutical preparation, transdermal transmission, injectionand epidermal administration. For transdermal transmission, absorptionpromoters or iontophoresis are suitable methods. Iontophoretictransmission may be accomplished using commercially available “patches”which deliver their product continuously via electric pulses throughunbroken skin for periods of several days or more.

Subjects Suitable for Treatment

The phrase “glucose metabolism disorder” encompasses any disordercharacterized by a clinical symptom or a combination of clinicalsymptoms that are associated with an elevated level of glucose and/or anelevated level of insulin in a subject relative to a healthy individual.Elevated levels of glucose and/or insulin may be manifested in thefollowing disorders and/or conditions: type II diabetes (e.g.insulin-resistance diabetes), gestational diabetes, insulin resistance,impaired glucose tolerance, hyperinsulinemia, impaired glucosemetabolism, pre-diabetes, metabolic disorders (such as metabolicsyndrome which is also referred to as syndrome X), obesity,obesity-related disorder; type I diabetes, idiopathic type 1 diabetes,latent autoimmune diabetes in adults, early-onset type 2 diabetes,youth-onset atypical diabetes, maturity onset diabetes of the young,malnutrition-related diabetes, and gestational diabetes.

An example of a suitable patient may be one who is hyperglycemic and/orhyperinsulinemic and who is also diagnosed with diabetes mellitus (e.g.Type II diabetes). “Diabetes” refers to a progressive disease ofcarbohydrate metabolism involving inadequate production or utilizationof insulin and is characterized by hyperglycemia and glycosuria.

“Hyperglycemia”, as used herein, is a condition in which an elevatedamount of glucose circulates in the blood plasma relative to a healthyindividual and can be diagnosed using methods known in the art. Forexample, hyperglycemia can be diagnosed as having a fasting bloodglucose level between 5.6 to 7 mM (pre-diabetes), or greater than 7 mM(diabetes).

“Hyperinsulinemia”, as used herein, is a condition in which there areelevated levels of circulating insulin while blood glucose levels mayeither be elevated or remain normal. Hyperinsulinemia can be caused byinsulin resistance which is associated with dyslipidemia such as hightriglycerides, high cholesterol, high low-density lipoprotein (LDL) andlow high-density lipoprotein (HDL), high uric acids, polycystic ovarysyndrome, type II diabetes and obesity. Hyperinsulinemia can bediagnosed as having a plasma insulin level higher than about 2 μU/mL.

A patient having any of the above disorders may be a suitable candidatein need of a therapy in accordance with the present method so as toreceive treatment for hyperglycemia, hyperinsulinemia, and/or glucoseintolerance. Administering the subject protein in such an individual canrestore glucose homeostasis and may also decrease one or more ofsymptoms associated with the disorder.

Candidates for treatment using the subject method may be determinedusing diagnostic methods known in the art, e.g. by assaying plasmaglucose and/or insulin levels. Candidates for treatment include thosewho have exhibited or are exhibiting higher than normal levels of plasmaglucose/insulin. Such patients include patients who have a fasting bloodglucose concentration (where the test is performed after 8 to 10 hourfast) of higher than about 100 mg/dL, e.g., higher than about 110 mg/dL,higher than about 120 mg/dL, about 150 mg/dL up to about 200 mg/dL ormore. Individuals suitable to be treated also include those who have a 2hour postprandial blood glucose concentration or a concentration after aglucose tolerance test (e.g. 2 hours after ingestion of a glucose-richdrink), in which the concentration is higher than about 140 mg/dL, e.g.,higher than about 150 mg/dL up to 200 mg/dL or more. Glucoseconcentration may also be presented in the units of mmol/L, which can beacquired by dividing mg/dL by a factor of 18.

Screening Methods

The present disclosure provides methods for identifying a candidateagent for reducing blood glucose levels and/or increasing insulinsensitivity in an individual. A subject screening method generallyinvolves: a) contacting a polypeptide comprising an intracellular domainof a p75^(NTR) (e.g., a p75^(NTR) ICD polypeptide) with: i) a GTPaseselected from Rab31 and Rab5; and ii) a test agent; and b) determiningthe effect of the test agent on binding of the intracellular domain ofp75^(NTR) to Rab31 or Rab5. In some cases, a subject screening method isa cell-free in vitro assay. In other cases, a subject screening methodis a cell-based in vitro assay. For example, a subject method cancomprise: a) contacting in a reaction mixture: i) a polypeptidecomprising an intracellular domain of a p75^(NTR) (e.g., a p75^(NTR) ICDpolypeptide); ii) a GTPase selected from Rab31 and Rab5; and iii) a testagent; and b) determining the effect of the test agent on binding of theintracellular domain of p75^(NTR) to Rab31 or Rab5.

In some cases, the polypeptide comprising an intracellular domain of ap75^(NTR) (e.g., a p75^(NTR) ICD polypeptide) is immobilized. In othercases, the GTPase is immobilized. In some cases, the Rab31 GTPasepolypeptide is a subject Rab31-derived peptide.

A test agent that inhibits binding of the intracellular domain ofp75^(NTR) to Rab31 or Rab5 is considered a candidate agent for reducingblood glucose levels and/or increasing insulin sensitivity. For example,a test agent that inhibits binding of the intracellular domain ofp75^(NTR) to Rab31 or Rab5 by at least about 15%, at least about 20%, atleast about 25%, at least about 30%, at least about 35%, at least about40%, at least about 50%, at least about 60%, at least about 70%, or morethan 70%, is considered a candidate agent for reducing blood glucoselevels and/or increasing insulin sensitivity.

As used herein, the term “determining” refers to both quantitative andqualitative determinations and as such, the term “determining” is usedinterchangeably herein with “assaying,” “measuring,” and the like.

The terms “candidate agent,” “test agent,” “agent,” “substance,” and“compound” are used interchangeably herein. Candidate agents encompassnumerous chemical classes, typically synthetic, semi-synthetic, ornaturally-occurring inorganic or organic molecules. Candidate agentsinclude those found in large libraries of synthetic or naturalcompounds. For example, synthetic compound libraries are commerciallyavailable from Maybridge Chemical Co. (Trevillet, Cornwall, UK),ComGenex (South San Francisco, Calif.), and MicroSource (New Milford,Conn.). A rare chemical library is available from Aldrich (Milwaukee,Wis.). Alternatively, libraries of natural compounds in the form ofbacterial, fungal, plant and animal extracts are available from Pan Labs(Bothell, Wash.) or are readily producible.

Candidate agents may be small organic or inorganic compounds having amolecular weight of more than 50 and less than about 10,000 daltons,e.g., a candidate agent may have a molecular weight of from about 50daltons to about 100 daltons, from about 100 daltons to about 150daltons, from about 150 daltons to about 200 daltons, from about 200daltons to about 500 daltons, from about 500 daltons to about 1000daltons, from about 1,000 daltons to about 2500 daltons, from about 2500daltons to about 5000 daltons, from about 5000 daltons to about 7500daltons, or from about 7500 daltons to about 10,000 daltons. Candidateagents may comprise functional groups necessary for structuralinteraction with proteins, particularly hydrogen bonding, and mayinclude at least an amine, carbonyl, hydroxyl or carboxyl group, and maycontain at least two of the functional chemical groups. The candidateagents may comprise cyclical carbon or heterocyclic structures and/oraromatic or polyaromatic structures substituted with one or more of theabove functional groups. Candidate agents are also found amongbiomolecules including peptides, saccharides, fatty acids, steroids,purines, pyrimidines, derivatives, structural analogs or combinationsthereof.

Assays of the present disclosure include controls, where suitablecontrols include a sample (e.g., a sample comprising a p75^(NTR) ICDpolypeptide, and a Rab5 or Rab31 polypeptide, in the absence of the testagent). Generally a plurality of assay mixtures is run in parallel withdifferent agent concentrations to obtain a differential response to thevarious concentrations. Typically, one of these concentrations serves asa negative control, i.e. at zero concentration or below the level ofdetection.

A variety of other reagents may be included in the screening assay.These include reagents such as salts, neutral proteins, e.g. albumin,detergents, etc., including agents that are used to facilitate optimalenzyme activity and/or reduce non-specific or background activity.Reagents that improve the efficiency of the assay, such as proteaseinhibitors, anti-microbial agents, etc. may be used. The components ofthe assay mixture are added in any order that provides for the requisiteactivity. Incubations are performed at any suitable temperature, e.g.,between 4° C. and 40° C. Incubation periods are selected for optimumactivity, but may also be optimized to facilitate rapid high-throughputscreening. Typically between 0.1 hour and 1 hour will be sufficient.

In some embodiments, a test compound of interest has an EC₅₀ of fromabout 1 nM to about 1 mM, e.g., from about 1 nM to about 10 nM, fromabout 10 nM to about 15 nM, from about 15 nM to about 25 nM, from about25 nM to about 50 nM, from about 50 nM to about 75 nM, from about 75 nMto about 100 nM, from about 100 nM to about 150 nM, from about 150 nM toabout 200 nM, from about 200 nM to about 250 nM, from about 250 nM toabout 300 nM, from about 300 nM to about 350 nM, from about 350 nM toabout 400 nM, from about 400 nM to about 450 nM, from about 450 nM toabout 500 nM, from about 500 nM to about 750 nM, from about 750 nM toabout 1 μM, from about 1 μM to about 10 μM, from about 10 μM to about 25μM, from about 25 μM to about 50 μM, from about 50 μM to about 75 μM,from about 75 μM to about 100 μM, from about 100 μM to about 250 μM,from about 250 μM to about 500 μM, or from about 500 μM to about 1 mM.

A candidate agent can be assessed for any cytotoxic activity it mayexhibit toward a living cell, using well-known assays, such as trypanblue dye exclusion, an MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)assay, and the like. Agents that do not exhibit cytotoxic activity areconsidered candidate agents.

Whether a test agent reduces p75^(NTR) ICD polypeptide binding to a Rab5or Rab31 polypeptide can be determined using any assay method suitablefor determining protein-protein binding. Such assays include, e.g.,enzyme-linked immunosorbent assays (ELISA); bioluminescence resonanceenergy transfer (BRET) assays; Förster resonance energy transfer (FRET)assays; immunoprecipitation assays; protein blot assays;radioimmunoassays; and the like.

In many embodiments, the screening method is carried out in vitro, in acell-free assay. In some embodiments, the in vitro cell-free assay willemploy a purified p75^(NTR) ICD polypeptide, where “purified” refers tofree of contaminants or any other undesired components. Purifiedp75^(NTR) ICD polypeptide that is suitable for a subject screeningmethod is at least about 50% pure, at least about 60% pure, at leastabout 70% pure, at least about 75% pure, at least about 80% pure, atleast about 85% pure, at least about 90% pure, at least about 95% pure,at least about 98% pure, at least about 99% pure, or greater than 99%pure. Similarly the Rab5 or Rab31 polypeptide can be purified.

Purified p75^(NTR) ICD polypeptide, and/or purified Rab5 or Rab31polypeptide, will in some embodiments be stabilized by addition of oneor more stabilizing agents, to maintain enzymatic activity. In someembodiments, a solution of purified p75^(NTR) ICD polypeptide, and/orpurified Rab5 or Rab31 polypeptide, comprises an aqueous solutioncomprising such polypeptide and from about 10% to about 50% glycerol,e.g., from about 10% to about 15%, from about 15% to about 20%, fromabout 20% to about 25%, from about 25% to about 30%, from about 30% toabout 35%, from about 35% to about 40%, from about 40% to about 45%, orfrom about 45% to about 50% glycerol. In some embodiments, a solutioncomprising a p75^(NTR) ICD polypeptide, and/or purified Rab5 or Rab31polypeptide, further comprises one or more of a chelating agent (e.g.,EDTA or EGTA); salts such as NaCl, MgCl₂, KCl, and the like; buffers,such as a Tris buffer, phosphate-buffered saline, sodium pyrophosphatebuffer, and the like; one or more protease inhibitors; and the like.

A p75^(NTR) ICD polypeptide suitable for use in a subject screeningmethod has a length of from about 15 amino acids to about 100 aminoacids, as set out above, and has at least about 90%, at least about 95%,at least about 97%, at least about 98%, at least about 99%, or 100%,amino acid sequence identity with a contiguous stretch of from about 15amino acids to about 20 amino acids, from about 20 amino acids to about25 amino acids, from about 25 amino acids to about 30 amino acids, fromabout 30 amino acids to about 35 amino acids, from about 35 amino acidsto about 40 amino acids, from about 40 amino acids to about 45 aminoacids, from about 45 amino acids to about 50 amino acids, from about 50amino acids to about 55 amino acids, from about 55 amino acids to about60 amino acids, from about 60 amino acids to about 65 amino acids, fromabout 65 amino acids to about 70 amino acids, from about 70 amino acidsto about 75 amino acids, from about 75 amino acids to about 80 aminoacids, from about 80 amino acids to about 85 amino acids, from about 85amino acids to about 90 amino acids, or from about 90 amino acids to 92amino acids or 95 amino acids, of an amino acid sequence depicted inFIG. 12 (i.e., one of SEQ ID NOs:46-50).

In some cases, the intracellular domain (ICD) of p75^(NTR) comprises theamino acid sequence LLASWATQDSATLDA (SEQ ID NO:1), or an amino acidsequence differing by no more than 1, 2, 3, 4, or 5 amino acids fromLLASWATQDSATLDA (SEQ ID NO:1), as long as the p75^(NTR) ICD polypeptidecan bind to the Rab5 or Rab31 polypeptide.

A Rab5 polypeptide suitable for use in a subject screening methodcomprises an amino acid sequence having least about 75%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity with a contiguousstretch of from about 150 amino acids to about 200 amino acids, or fromabout 200 amino acids to about 215 amino acids, of the amino acidsequence depicted in FIG. 13 (SEQ ID NO:51).

A Rab31 polypeptide suitable for use in a subject screening methodcomprises an amino acid sequence having least about 75%, at least about85%, at least about 90%, at least about 95%, at least about 98%, atleast about 99%, or 100%, amino acid sequence identity with a contiguousstretch of from about 125 amino acids to about 150 amino acids, fromabout 150 amino acids to about 175 amino acids, or from about 175 aminoacids to about 195 amino acids, with the amino acid sequence depicted inFIG. 14 (SEQ ID NO:52).

In some embodiments, a subject assay will employ a fusion protein,comprising a p75^(NTR) ICD polypeptide fused in-frame to a fusionpartner. In some embodiments, the fusion partner is attached to theamino terminus of the p75^(NTR) ICD polypeptide. In other embodiments,the fusion partner is attached to the carboxyl terminus of the p75^(NTR)ICD polypeptide. In other embodiments, the fusion partner is fusedin-frame to the p75^(NTR) ICD polypeptide at a location internal to thep75^(NTR) ICD polypeptide. Suitable fusion partners includeimmunological tags such as epitope tags, including, but not limited to,hemagglutinin, FLAG, and the like; proteins that provide for adetectable signal, including, but not limited to, fluorescent proteins,enzymes (e.g., β-galactosidase, luciferase, horse radish peroxidase,etc.), and the like; polypeptides that facilitate purification orisolation of the fusion protein, e.g., metal ion binding polypeptidessuch as 6His tags (e.g., p75^(NTR) ICD/6His), glutathione-S-transferase,and the like; polypeptides that provide for subcellular localization;and polypeptides that provide for secretion from a cell.

In some embodiments, the fusion partner is an epitope tag. In someembodiments, the fusion partner is a metal chelating peptide. In someembodiments, the metal chelating peptide is a histidine multimer, e.g.,(His)₆. In some embodiments, a (His)₆ multimer is fused to the aminoterminus of a p75^(NTR) ICD polypeptide; in other embodiments, a (His)₆multimer is fused to the carboxyl terminus of a p75^(NTR) ICDpolypeptide. The (His)₆-p75^(NTR) ICD fusion protein is purified usingany of a variety of available nickel affinity columns (e.g. His-bindresin, Novagen).

In some embodiments, a subject screening method is carried out in vitroin a cell, e.g., a cell grown in cell culture as a unicellular entity.Suitable cells include, e.g., eukaryotic cells, e.g., mammalian cellssuch as CHO cells 293 cells, 3R3 cells, and the like.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake and use the present invention, and are not intended to limit thescope of what the inventors regard as their invention nor are theyintended to represent that the experiments below are all or the onlyexperiments performed. Efforts have been made to ensure accuracy withrespect to numbers used (e.g. amounts, temperature, etc.) but someexperimental errors and deviations should be accounted for. Unlessindicated otherwise, parts are parts by weight, molecular weight isweight average molecular weight, temperature is in degrees Celsius, andpressure is at or near atmospheric. Standard abbreviations may be used,e.g., bp, base pair(s); kb, kilobase(s); pl, picoliter(s); s or sec,second(s); min, minute(s); h or hr, hour(s); aa, amino acid(s); kb,kilobase(s); bp, base pair(s); nt, nucleotide(s); i.m.,intramuscular(ly); i.p., intraperitoneal(ly); s.c., subcutaneous(ly);and the like.

Example 1 p75 Neurotrophin Receptor Regulates Glucose Homeostasis andInsulin Sensitivity

Materials and Methods

Mice.

p75^(NTR−/−) mice (Lee et al. (1992) Cell 69:737-749) were obtained fromJackson Laboratory. Mice were on a C57BL/6 background and were furthercrossed to C57BL/6 background (11-12 crossings). Heterozygousp75^(NTR+/−) were crossed to obtain p75^(NTR−/−, p)75^(NTR+/−) andp75^(NTR+/+) littermates used in experiments. Male mice were fed withstandard chow and had access to food and water ad libitum. MRIexperiments were carried out on a Varian 7T 300 MHz Horizontal Bore MRISystem at the Department of Radiology & Biomedical Imaging at UCSF. Fatand lean tissue volumes were assessed by using the Volocity 3D ImageAnalysis Software (PerkinElmer). All animal study protocols werereviewed and approved by the Institutional Animal Care and Use Committeeof the University of California San Francisco and San Diego and are inaccordance with those set by the National Institutes of Health.

Glucose and Insulin Tolerance Tests.

Glucose and insulin tolerance tests (GTT and ITT) were performed on 6 hfasted mice. For GTT, animals were injected i.p. with dextrose (1 g/Kg,Hospira, Inc) whereas for ITT 0.4 units/Kg insulin (Novolin R,Novo-Nordisk) was injected i.p. Glucose excursion following injectionwas monitored over time. Blood samples were drawn at 0, 10, 30, 60 and120 min after dextrose or insulin injection. Glucose was measured usinga One-touch glucose-monitoring system (Lifescan).

Hyperinsulinemic-Euglycemic Clamp Studies.

Clamp studies were performed as previously described (2-4). Briefly,dual jugular catheters were implanted and mice were allowed to recoverthree days before clamp procedure. Following 6 h fast, an equilibratingsolution (5.0 μCi/h, 0.12 ml/h [3-³H]D-glucose, NEN Life ScienceProducts) was infused into one of the jugular catheters for 90 min toequilibrate the plasma tracer. Following this equilibrating period, abasal blood sample was drawn via tail vein to obtain tracer counts forthe calculation of basal glucose uptake. The insulin (8 mU/Kg/min, at aflow rate of 2 ml/min) plus tracer (0.5 μCi/h) infusion and glucose (50%dextrose, Abbot, variable rate) were initiated simultaneously, with theglucose flow rate adjusted to reach a steady state blood glucoseconcentration (˜120 min). Steady state was confirmed by stable tracercounts in plasma during final 30 min of clamp. At the end of the clamp,a blood sample was taken at 110 and 120 min for the determination oftracer-specific activity. And at the steady state, the rate of glucosedisappearance or the total glucose disposal rate (GDR) is equal to thesum of the rate of endogenous or hepatic glucose production (HGP) plusthe exogenous glucose infusion rate (GIR). The insulin-stimulatedglucose disposal rate (IS-GDR) is equal to the total GDR minus the basalglucose turnover rate.

Cell Culture and Primary Adipocyte Isolation.

3T3L1 cells were maintained in DMEM containing 10% bovine serum. Allmedia were supplemented with 1% penicillin/streptomycin (Invitrogen).Mouse embryonic fibroblasts (MEFs) were isolated from wild-type (WT) andp75^(NTR−/−) embryos as described (5). Briefly, heads and internalorgans were removed from E12-14 embryos, and the remaining embryo wasminced with scissors. Ten ml of ice cold phosphate-buffered saline (PBS)was added and minced embryos were centrifuged for 5 minutes at 500×g.Each embryo was resuspended in 7.5 ml Trypsin-EDTA for 20 minutes at 37°C. with occasional shaking. After trypsin deactivation by 7.5 ml DMEMwith 10% FBS the suspension was poured through a 70 μm filter and cellswere cultured in DMEM with 10% fetal bovine serum (FBS). Differentiationof 3T3L1 cells and MEFs was performed as described (6). Briefly,differentiation was induced by incubating confluent monolayers for 3days in DMEM containing 10% fetal bovine serum (FBS), 0.5 mM3-isobutyl-1-methylxanthine, 0.4 μg/ml dexamethasone, and 1 μg/ml ofinsulin. Medium was replaced every 2 days with DMEM containing 10% FBS.After 8-12 days, 75% of the cells expressed the adipocyte phenotype asassessed by Oil Red O. Isolation of primary adipocytes from epididymalfat pads was performed as described (7). Briefly, fat pads were mincedin Krebs-Ringer Bicarbonate-Hepes (KRBH) buffer, pH 7.4, treated withcollagenase (Invitrogen) for 40 min, filtered through a nylon strainerand centrifuged at 400×g for 1 minute. Fully-differentiated adipocyteswere transfected by electroporation using Amaxa Cell Line Nucleofectorkit L (Lonza) following manufacturer's instructions. For p75^(NTR)knockdown experiments, 3T3L1 cells were differentiated for 6-8 days andinfected with lentivirus encoding a short hairpin shRNA for mousep75^(NTR) (8) and L6 myocytes were transfected with siGENOME SMART poolfor rat p75^(NTR) (Thermo Scientific).

Glucose Uptake Assay.

Fully differentiated 3T3L1 adipocytes and eight day differentiated WTand p75^(NTR−/−) MEFs were serum-starved for 3 hrs in DMEM and thenequilibrated in Hepes/Salt (H/S) buffer for 30 min prior to treatmentwith 100 nM insulin for 30 min at 37° C. Thirty min after the additionof insulin, [³H]-2-deoxyglucose (Amersham) and 2 mM 2-deoxyglucose(Sigma) in H/S buffer was added. After incubation at 37° C. for 10 min,the reactions were terminated by washing twice with ice-cold PBS. Cellswere lysed in 1 M NaOH for 30 min at room temperature. An equal volumeof 1 M HCl was added and incorporated radioactivity was measured byliquid scintillation counting. 3T3L1 adipocytes and WT and p75^(NTR−/−)MEF-derived adipocytes were transfected by electroporation with greenfluorescent protein (GFP), p75^(NTR) FL, and p75^(NTR) intracellulardomain (ICD); and Rab5S34N and Rab31Q64L expression vectors were alsoused. For neurotrophin stimulation, 3T3L1 adipocytes were treated with100 ng/ml of nerve growth factor (NGF) (Peprotech) or 50 ng/ml ofbrain-derived neurotrophic factor (BDNF) (Peprotech) for 1 h or 12 hbefore insulin stimulation. Measurement of glucose uptake in primaryadipocytes was performed as described (9), after several washes withKRBH buffer, a 25-30% suspension of primary isolated adipocytes wasincubated with KRBH buffer without glucose for 30 min prior to insulinstimulation. After insulin stimulation 0.1 mM [³H]-2DG (Perkin Elmer)were added and incubated for 10 min. Immediately after incubation, thesuspension was loaded on top of 0.2 ml of silicon oil in a 0.4 mlpolyethylene tube and centrifuged. The fat cells were isolated and thelevel of radioactivity was counted. An aliquot of the suspension wasused to determine protein concentration.

Cell Fractionation, Western Blotting and Antibodies.

For fractionation experiments, cells were washed twice with 20 mM TrisHCl/1 mM EDTA/255 mM sucrose, pH 7.4, then resuspended and homogenizedwith 10 strokes of a pestle. Cell lysates were centrifuged at 4° C. at16000 g for 15 min. The pellet was resuspended in buffer, spun again,and the new pellet was resuspended in buffer, and was applied to a 1.12M sucrose cushion containing 20 mM Tris HCl and 1 mM EDTA andcentrifuged at 101,000×g for 70 min. Plasma membranes were collectedfrom the interface and resuspended in buffer. Equal amounts of proteinwere then analyzed by Western blotting as described (8). Antibodies usedwere: mouse anti-Glut4, 1:1000, (Abcam); mouse anti-Glut1, 1:1000,(Abcam); rabbit anti-Ser 473 phospho-Akt, 1:1000, (Cell signaling);rabbit anti-Akt, 1:1000, (Cell signaling); mouse anti-Rab31, 1:1000,(Abnova); rabbit anti-Rab5, 1:1000, (Abcam); rabbit anti-myc, 1:1000,(Abcam); mouse anti-HA, 1:1000, (Cell Signaling); rabbit anti-GFP,1:1000, (Santa Cruz Biotechnology); rabbit anti-p75^(NTR) 9992, 1:2000;rabbit anti-p75^(NTR), 1:1000 (Millipore); rabbit anti-Actin, 1:1000,(Sigma); rabbit anti-Gapdh, 1:3000, (Abcam). Quantification wasperformed on the Scion NIH Imaging Software.

Co-Immunoprecipitation (Co-IP).

Co-IP was performed as previously described (22). Briefly, cell lysateswere prepared in 1% Nonidet P-40 (NP-40; non-ionic detergent), 200 mMNaCl, 1 mM EDTA, and 20 mM Tris-HCl, pH 8.0. Immunoprecipitations (IPs)were performed with an anti-p75^(NTR) antibody and immunoblot withanti-Rab31 and anti-p75^(NTR). For mapping experiments, myc-Rab31 cDNAwas cotransfected with HA-tagged p75^(NTR) deletion constructs intoHEK293T cells. Immunoprecipitation (IP) was performed with an anti-mycantibody. Cell lysates were probed with an anti-hemagglutinin (anti-HA)or an anti-myc antibody. For co-IP experiments using recombinantproteins, equimolar amounts (1 μM) of purified recombinant glutathione-Stransferase (GST), activated GST-Rab5, and p75^(NTR) ICD were mixed inbinding buffer (5% glycerol, 20 mM Tris-HCl pH=7.5, 100 mM NaCl, 10 mMEDTA, 0.025% Tween-20) and incubated for 1 hour at 4° C. To nucleotideload GST-Rab5 recombinant protein, GST-Rab5 was incubated with 5%glycerol, 20 mM Tris-HCl pH=7.5, 150 mM NaCl, 5 mM EDTA, containing 1 mMGTPγS at 30° C. for 30 min. The reaction was stopped by adding 60 mMMgCl₂. For inhibition experiments, 1 μM Tat-Pep5 (Calbiochem) or 1 μMcontrol Tat-fused peptide (Calbiochem) were added for 1 h together withthe recombinant p75ICD and activated GST-Rab5. Washedglutathione-sepharose beads were added according to the manufacturer'sinstructions for an additional hour. Beads were sedimented bycentrifugation (10000×g for 1 min) and washed three times. Proteinsassociated with the beads were eluted by boiling in loading buffer andseparated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE).

In Vitro GST Activity Assay.

The activity assay was performed as described (10) in serum-starved WTand p75^(NTR−/−) MEF-derived adipocytes treated with insulin asindicated, 1 mg of GST-EEA1/ml PBS was incubated with 1 ml GSH sepharosebeads for 1 h at 4° C. Beads were spun down, washed 10 times, andresuspended in PBS to create a 50% slurry. 2 mg of lysate in a finalvolume of 500 ml lysis buffer were immunoprecipitated with 50 μl ofGST-EEA1-bound GSH sepharose bead slurry overnight at 4° C. to pull downthe active forms of Rab5 and Rab31. Beads were spun down, washed, boiledin sodium dodecyl sulfate (SDS), and loaded on 8-12% Tris-Glycine geland blotted for Rab5 or Rab31. Whole cell lysates taken beforeincubation with beads was used for Western blotting for Rab5, Rab31 andActin.

Peptide Array Mapping.

Peptide arrays were manufactured by Kinexus Bioinformatics Corporation(Vancouver, Canada). Synthetic overlapping peptides (25 amino acids inlength) for p75ICD were spotted on nitrocellulose membranes. Membraneswere overlaid with 1 μg/ml recombinant GST-Rab31, GST-Rab5 and p75^(NTR)ICD. Bound recombinant GST-Rab31 and GST-Rab5 were detected using rabbitanti-GST (1:1000, Cell Signaling) and bound recombinant p75ICD wasdetected using rabbit anti-p75^(NTR) (1:1000, Millipore) followed bysecondary anti-rabbit horseradish peroxidase antibody (1:5000, CellSignaling).

Immunocytochemistry.

p75^(NTR+/+) and p75^(NTR−/−) MEFs-derived adipocytes were transfectedby electroporation with the GFP-Glut4 expression vector. After 24 hours,adipocytes were serum starved for 2 hours in DMEM and stimulated with100 nM insulin for 30 min, fixed with paraformaldehyde and analyzed.Fully differentiated 3T3L1 were fixed with paraformaldehyde, and stainedusing the following antibodies: rabbit anti-p75^(NTR) (Millipore), mouseanti-Rab31 (Abnova) and rabbit anti-Rab5 (Abcam).

Microscopy and Image Acquisition.

Microscopic images were acquired with an Axioplan II epifluorescencemicroscope (Carl Zeiss, Inc.) and dry Plan-Neofluar objectives (10×0.3NA, 20×0.5 NA, or 40×0.75 NA) using an Axiocam HRc CCD camera and theAxiovision image analysis software.

Statistical Analysis.

Data are shown as the means±SEM. Differences between groups wereexamined by unpaired Student's t test or by one-way ANOVA for multiplecomparisons followed by Bonferroni's correction for comparison of means.

Results

Loss of p75^(NTR) Increases Insulin Sensitivity Independently of BodyWeight.

On normal chow, p75^(NTR−/−) mice weigh the same as wild-type (WT) mice(FIG. 1A and FIG. 2A), consume the same amount of food (FIG. 2B), andshow similar body composition (FIG. 26), suggesting that loss ofp75^(NTR) does not regulate appetite or body weight on normal diet. Todetermine if p75^(NTR) regulates other metabolic pathways, glucose andinsulin homeostasis were tested in WT and p75^(NTR−/−) mice.p75^(NTR−/−) mice showed lowered glycemic excursions during glucosetolerance tests (GTTs) (50% decrease in AUC) (FIG. 1B) with a 30%increase in the hypoglycemic effect of insulin during insulin tolerancetests (ITT) (FIGS. 1C and D) compared to WT controls. These findingsindicate that lean, chow-fed p75^(NTR−/−) mice are insulin sensitiverelative to weight-matched lean controls, and this was quantitated byeuglycemic-hyperinsulinemic glucose clamp studies. As seen in FIG. 1E,the glucose disposal rate (GDR) was greater in p75^(NTR−/−) mice, as wasthe insulin stimulated GDR (IS-GDR) value (FIG. 1F) compared to WT mice.Basal hepatic glucose production (HGP) was not affected by p75^(NTR)deficiency, but suppression of HGP by insulin was significantly improved(FIG. 1G) by 20% (FIG. 1H). Furthermore, free fatty acid (FFA) levelswere lower in the p75^(NTR−/−) mice during the clamp studies, whilebasal FFA, glucose and insulin levels were unchanged (FIG. 3A-C). It isimportant to note that genetic manipulations that improve insulinsensitivity in lean, normal chow-fed mice are unusual, and these resultsdemonstrate significantly enhanced insulin sensitivity in p75^(NTR−/−)mice independent of food intake or body weight.

FIG. 1.

p75^(NTR−/−) mice exhibit improved insulin sensitivity on normal diet.(A) Body weight of WT and p75^(NTR−/−) mice on normal chow (n=10; ns,not significant). (B) Glucose tolerance of fasted WT and p75^(NTR−/−)mice on normal chow (n=10, *P<0.05, **P<0.01, ***P<0.001). (C) Insulintolerance of fasted WT and p75^(NTR−/−) mice on normal chow (n=10,**P<0.01, ***P<0.001). (D) Percentage of initial blood glucose ininsulin tolerance test (n=10, *P<0.05). (E) Glucose disposal rates (GDR)from WT and p75^(NTR−/−) mice on normal chow (n=10, **P<0.01). (F)Insulin-stimulated glucose disposal rates (IS-GDR) of WT andp75^(NTR−/−) mice on normal chow (n=10, *P<0.05). (G) Hepatic glucoseproduction (HGP) from WT and p75^(NTR−/−) mice on normal chow (n=10,*P<0.05). (H) Suppression of HGP in WT and p75^(NTR−/−) mice on normalchow (n=10, *P<0.05). Data are shown as the means±SEM. Statisticalcomparisons between means were made with one-way ANOVA (B, C and D) andStudent's t test (E, F, G and H).

FIG. 2.

Metabolic analysis of p75^(NTR−/−) mice on normal chow. (A) Body weighton normal chow of p75^(NTR+/+) (n=4), p75^(NTR+/−) (n=5) andp75^(NTR−/−) (n=7) mice. (*P<0.05). Data are shown as the means±SEM.Statistical comparisons between means were made with one-way ANOVA. (B)Food consumption on normal chow of WT and p75^(NTR−/−) mice (n=5). (C)MRI images from 10 week old WT (n=4) and p75^(NTR−/−) mice (n=5) onnormal chow (left). Fat and lean tissue volumes (cm³) from the WT andp75^(NTR−/−) mice analyzed by MRI (ns, not significant by Student's ttest) (right).

FIG. 3.

(A) Basal levels of FFA after clamp experiments in WT and p75^(NTR−/−)mice (n=10) (ns, not significant; *P<0.05). (B) Basal levels of bloodglucose after clamp experiments in WT and p75^(NTR−/−) mice (n=10) (ns,not significant). (C) Basal levels of insulin after clamp experiments inWT and p75^(NTR−/−) mice (n=10) (ns, not significant). “Basal” refers tothe values for each of these measures after an overnight fast. “Clamp”refers to the values for each of these measures after the clamp period.(D) Western blot analysis for p75^(NTR) in MEF-derived adipocytes, WATand skeletal muscle. Representative blot from three independentexperiments is shown. Data are shown as the means±SEM. Statisticalcomparisons between means were made with one-way ANOVA.

Loss of p75^(NTR) Increases Glucose Uptake.

The mechanisms by which p75^(NTR) might regulate insulin sensitivity incell autonomous in vitro systems were explored. Since p75^(NTR−/−) micedisplayed increased in vivo glucose disposal (FIG. 1E), the effects ofp75^(NTR) on insulin-stimulated glucose uptake was examined inadipocytes and myocytes. Adipocytes derived from p75^(NTR−/−) mouseembryonic fibroblasts (MEFs) showed a 3-fold increase ininsulin-stimulated glucose uptake compared to WT (FIG. 4A), while basalglucose uptake was identical between WT and p75^(NTR−/−) MEF-derivedadipocytes (FIG. 4A). Similar differences in insulin-stimulated glucosetransport were observed in primary adipocytes isolated from WT andp75^(NTR−/−) mice (FIG. 4B). Likewise, shRNA knockdown of p75^(NTR)increased insulin-stimulated glucose uptake in 3T3L1 adipocytes (FIG.46). To analyze the potential role of p75^(NTR) in skeletal muscle, theinfluence of p75^(NTR) on insulin-stimulated glucose uptake was examinedin L6 myocytes. Similar to adipocytes (FIG. 4C), L6 cells transfectedwith p75^(NTR) siRNA showed no change in basal glucose transport butdisplayed a significant 20% increase in insulin-stimulated glucoseuptake compared to cells transfected with a control siRNA (FIG. 4D).Efficiency of the p75^(NTR) knockdown in 3T3L1 adipocytes and L6myoblasts (FIG. 4C, D), and expression of p75^(NTR) in MEF-derivedadipocytes, WAT and skeletal muscle (FIG. 3D) is shown by immunoblot.

Consistent with the results of p75^(NTR) depletion, overexpression offull-length p75^(NTR) (p75FL) rescued the increase in glucose uptake in75^(NTR−/−) adipocytes (FIG. 4E). The intracellular domain (ICD) ofp75^(NTR), which lacks the extracellular domain that bindsneurotrophins, was also overexpressed. Similar to the p75FL, the p75ICDalso rescued the increase in glucose uptake in p75^(NTR−/−) adipocytes(FIG. 4E), while p75FL and p75ICD decreased glucose uptake in WTadipocytes (FIG. 4E, 5A). These results indicate that the ability ofp75^(NTR) to regulate glucose uptake is neurotrophin independent.Consistent with this, addition of the p75^(NTR) neurotrophin ligandsNerve Growth Factor (NGF) or Brain Derived Neurotrophin Factor (BDNF)did not affect glucose uptake (FIG. 5B). Together, these resultsindicate that p75^(NTR) regulates glucose uptake in adipocytes andmyoblasts, and are consistent with the improved in vivo insulinsensitivity and glucose disposal in p75^(NTR−/−) mice (FIG. 1).

FIG. 4.

p75^(NTR) regulates glucose uptake in adipocytes and skeletal musclecells. (A) Basal and insulin-stimulated glucose uptake in WT andp75^(NTR−/−) MEF-derived adipocytes (ns, not significant; *P<0.05). (B)Basal and insulin-stimulated glucose uptake in WT and p75^(NTR−/−)primary isolated adipocytes (ns, not significant; *P<0.05). (C)p75^(NTR) expression in 3T3L1 adipocytes infected with p75^(NTR) shRNAor scrambled shRNA lentivirus. Gapdh was used as a loading control(top). Basal and insulin-stimulated glucose uptake in 3T3L1 cellsdifferentiated to adipocytes and infected with lentivirus containingp75^(NTR) shRNA or scrambled shRNA control (*P<0.05; **P<0.01) (bottom).(D) p75^(NTR) expression in L6 myocytes tranfected with p75^(NTR) siRNAor siRNA control. Gapdh was used as a loading control (top). Basal andinsulin-stimulated glucose uptake in L6 myocytes cells transfected withp75^(NTR) siRNA or siRNA control (**P<0.01; ***P<0.001) (bottom). (E)Basal and insulin-stimulated glucose uptake in WT and p75^(NTR−/−)MEF-derived adipocytes electroporated with p75FL, p75ICD or GFP controlexpression vectors. After 72 h, cells were serum starved for 3 h andthen stimulated with 100 nM insulin (*P<0.05). Data are shown as themeans±SEM of a minimum of three different experiments performed intriplicate. Statistical comparisons between means were made with one-wayANOVA (A, B and E) and Student's t test (C and D).

FIG. 5. (A) Basal and insulin-stimulated glucose uptake in 3T3L1adipocytes electroporated with p75^(NTR) FL expression vector or GFPcontrol vector. After 72 h, cells were serum starved for 3 h and thenstimulated with or without insulin (ns, not significant; *P<0.05). (B)Basal and insulin-stimulated glucose uptake in 3T3L1 adipocytes treatedwith neurotrophins. No statistically significant differences wereobserved between basal and insulin-stimulated glucose uptake in 3T3L1adipocytes treated with NGF or BDNF for 1 h before insulin stimulation.Similar results were obtained after 12 h neurotrophin treatment (notshown). Data are shown as the means±SEM of a minimum of three differentexperiments performed in triplicate. Statistical comparisons betweenmeans were made with one-way ANOVA.

p75^(NTR) Regulates GLUT4 Retention.

Since regulation of insulin-stimulated glucose transport is a novelcellular function for p75^(NTR), studies were undertaken to determinethe molecular mechanism downstream of p75^(NTR) that regulates glucoseuptake. Insulin-stimulated glucose uptake is largely due totranslocation of GLUT4 from its basal intracellular location to theplasma membrane (4, 24). To determine if p75^(NTR) regulates GLUT4trafficking, the intracellular localization of GLUT4-GFP was examined inp75^(NTR−/−) adipocytes. In response to insulin, p75^(NTR−/−) adipocytesexhibited increased GLUT4 translocation to the plasma membrane comparedto WT adipocytes (FIG. 6A). Plasma membrane fractionation was alsoperformed from WAT derived from WT and p75^(NTR−/−) mice injected witheither saline or glucose to assess translocation of GLUT4 in response toendogenous insulin. As shown in FIG. 6B, plasma membrane GLUT4 contentwas increased in p75^(NTR−/−) WAT. p75^(NTR) deletion did not affecttotal protein levels of GLUT1 or GLUT4 in 3T3L1 adipocytes, MEF-derivedadipocytes, or primary adipocytes (FIG. 7A-C). Protein levels ofp75^(NTR) were shown by immunoblot (FIG. 7A-C). p75^(NTR) also did notaffect insulin receptor signaling, as loss of p75^(NTR) did not increaseinsulin-stimulated Akt phosphorylation (FIG. 7D).

FIG. 6.

p75^(NTR) regulates GLUT4 trafficking in adipocytes. (A)Immunocytochemistry of WT and p75^(NTR−/−) MEF-derived adipocytestransfected with GFP-tagged GLUT4 and stimulated with 100 nM of insulinfor 30 min. Scale bar, 75 μm. Representative images of two independentexperiments are shown (left). GLUT4 plasma membrane (PM) translocationwas quantified by determining the percentage of cells with GFP rimstaining. At least 50 cells per condition were counted (*P<0.05 byStudent's t test). (B) Western blot for GLUT4 in the plasma membranefraction of epididymal WAT from WT and p75^(NTR−/−) mice on normal chowinjected with (1 g/Kg) of dextrose after 6 h fasting. Ponceau red wasused as a loading control.

FIG. 7.

(A) Western blot for Glut1, Glut4 and p75^(NTR) in 3T3L1 adipocytesinfected with p75^(NTR) shRNA or scrambled shRNA control containinglentivirus. (B) Western blot for Glut1, Glut4 and p75^(NTR) in WT andp75^(NTR−/−) MEF-derived adipocytes. (C) Western blot for Glut1, Glut4and p75^(NTR) in primary isolated adipocytes. Gapdh was used as loadingcontrol. (D) Western blot for phospho-Akt in WT and p75^(NTR−/−) primaryadipocytes. Total Akt was used as a loading control. Representativeblots of three independent experiments are shown.

p75^(NTR) Regulates Rab5 and Rab31 Activities.

Stimulation of GLUT4 translocation by insulin results from traffickingof GLUT4 vesicles from intracellular storage sites, and their subsequentfusion with the plasma membrane (4). Activation of Rab5 GTPase functionsin the initial endocytosis of GLUT4, thus regulating the intracellularpools of GLUT4 (25). The intracellular retention of GLUT4 is sustainedby the activity of Rab31, a Rab5 subfamily GTPase implicated intrans-Golgi network-to-endosome trafficking (6). Insulin decreases theactivity of Rab31 in adipocytes, and Rab31 knockdown potentiatesinsulin-stimulated movement of GLUT4 to the cell surface with increasedglucose uptake (6). Since p75^(NTR) is known to regulate other GTPases,such as Rac (23), Ras (21), and RhoA (22), studies were undertaken todetermine if p75^(NTR) is required for activation of Rab5 and Rab31.EEA1 binds to Rab5 and Rab31 in a GTP-dependent manner (6). A pulldownassay with EEA1 showed increased activity of Rab5 and decreased activityof Rab31 in p75^(NTR−/−) adipocytes (FIG. 8A), suggesting that loss ofp75^(NTR) increases GLUT4 endocytosis, while decreasing GLUT4intracellular retention. Overexpression of dominant negative Rab5, butnot dominant active Rab31, rescued the increased insulin-induced glucoseuptake in the p75^(NTR−/−) adipocytes (FIG. 8B). These results suggestthat loss of p75^(NTR) differentially regulates Rab5 and Rab31 toincrease the flux of GLUT4 through the recycling pathway, whiledecreasing GLUT4 intracellular retention; thus resulting in increasedplasma membrane GLUT4 translocation and glucose uptake.

FIG. 8.

p75^(NTR) regulates Rab5 and Rab31 GTPase activity in adipocytes. (A) WTand p75^(NTR−/−) MEF-derived adipocytes were stimulated with insulin for5 and 20 min. The activation state of Rab31 and Rab5 was determinedusing GST-EEA1/NT. Rab31-GTP and Rab5-GTP levels normalized to Rab31 andRab5, respectively, were quantified by densitometry (Δ represents meanvalue). Representative blot of three independent experiments is shown.(B) Basal and insulin-stimulated glucose uptake in WT and p75^(NTR−/−)MEF-derived adipocytes electroporated with Rab31Q69L, Rab5S34N or GFPcontrol expression vector. After 72 h, cells were serum starved for 3 hand then stimulated with insulin (*P<0.05 vs WT basal; ^(#)P<0.05 vsp75^(NTR−/−) basal). Data are shown as the means±SEM of a minimum ofthree different experiments performed in triplicate. Statisticalcomparisons between means were made with one-way ANOVA.

the Rab5 Family GTPases Rab5 and Rab31 Directly Associate with the DeathDomain of p75^(NTR).

ICD of p75^(NTR) interacts with several binding partners to regulatecellular functions (26). Co-immunoprecipitation showed interaction ofp75^(NTR) with Rab31 (FIG. 9A) and Rab 5 (FIG. 5B). p75^(NTR) alsointeracted with Gapex5 (FIG. 10A), a Guanine nucleotide Exchange Factor(GEF) for Rab5 and Rab31 (6). Endogenous co-immunoprecipitation showedthat p75^(NTR) interacts with Rab31 and Rab5 in 3T3L1 adipocytes (FIG.9C). p75^(NTR) co-localized with Rab31 and Rab5 in 3T3L1 adipocytes(FIG. 10B). To evaluate the subcellular distribution of p75^(NTR) andRab5, subcellular fractions of adipocyte were analyzed. The majority ofp75^(NTR) protein in adipocytes was localized together with Rab5 inlow-density microsomes (LDMs) (FIG. 10C). This is in accordance withprior studies demonstrating that a majority of Rab5 resides in LDM (27).To verify the specificity of the association of p75^(NTR) with Rab5family GTPases, mapping studies were conducted using deletion mutants(FIG. 9D). Rab5 and Rab31 interact with p75FL, but not p75Δ83, adeletion missing the distal 83 amino acids (FIG. 9E, F), suggesting thatthe interaction between p75^(NTR) and Rab5 family GTPases occurs withinthe death domain (DD) of p75^(NTR). As expected, the deletion mutantp75Δ151, which lacks the entire p75ICD, also did not interact with Rab5and Rab31 (FIG. 9E, F). Overexpression of p75FL suppressedinsulin-stimulated glucose uptake, but not p75Δ83 (FIG. 9G), furthersupporting the involvement of the DD of p75^(NTR) in the regulation ofglucose uptake by p75^(NTR).

To further characterize the interaction, peptide array technology, whichhas defined sites of direct interaction for other p75^(NTR) partners(16, 18) and many other proteins (28), was used. Using Rab5-GST orRab31-GST, a peptide array library of overlapping 25-mer peptides thatspanned the sequence of p75ICD was screened. The strongest interactionswere within the DD and in particular within helix 4 and the regionbetween helixes 4 and 5 (peptide 7, aa 386-400) (FIG. 9I). Thisinteraction was distinct from that previously described for binding ofRhoGDI within helix 5 of p75ICD (FIG. 10D) (20). In accordance,Tat-Pep5, an inhibitory peptide that blocks the interaction of p75^(NTR)with RhoGDI (20), did not block the interaction of Rab5 with p75ICD(FIG. 9I), further suggesting that different epitopes within the DD ofp75^(NTR) contribute to the binding of Rho and the Rab5 family GTPases.These results suggest a direct interaction between p75^(NTR) and theRab5 family GTPases that primarily requires helix 4 within the deathdomain of p75^(NTR).

FIG. 9. The death domain of p75^(NTR) directly associates with Rab5 andRab31 GTPases. (A) Immunoprecipitation of HA-FL p75^(NTR) with myc-Rab31transfected in HEK293T cells. (B) Immunoprecipitation of HA-FL-p75^(NTR)with GFP-Rab5 transfected in HEK293T cells. Lysates wereimmunoprecipitated with anti-HA antibody, and western blots weredeveloped with anti-GFP and anti-HA antibodies. (C) Immunoprecipitationof p75^(NTR) with Rab31 and Rab5 in 3T3L1 adipocytes stimulated withinsulin. Lysates were immunoprecipitated with anti-p75^(NTR) antibody,and western blots were developed with anti-Rab31 and anti-Rab5antibodies. (D) Schematic diagram of HA-tagged p75FL and deletionmutants Δ83 and Δ151. TM, transmembrane domain; JX, juxtamembraneregion; DD, death domain. (E) Mapping of the p75^(NTR) sites requiredfor interaction with Rab31. Immunoprecipitation of myc-Rab31 withtruncated forms of HA-p75^(NTR) transfected in HEK293T cells. Lysateswere immunoprecipitated with anti-myc antibody, and western blots weredeveloped with anti-HA and anti-myc antibodies. (F) Mapping of thep75^(NTR) sites required for interaction with Rab5. Immunoprecipitationof truncated forms of HA-p75^(NTR) with GFP-Rab5 in HEK293T cells.Lysates were immunoprecipitated with anti-HA antibody, and western blotswere developed with anti-GFP and anti-HA antibodies. (G) Basal andinsulin-stimulated glucose uptake of WT MEF-derived adipocyteselectroporated with p75FL, p75Δ83 or GFP control expression vectors.After 72 h, cells were serum starved for 3 h and then stimulated with100 nM insulin (**P<0.01; ***P<0.001; ns, not significant). Data areshown as the means±SEM of two different experiments performed intriplicate. Statistical comparisons between means were made with one-wayANOVA. (H) Peptide array mapping of the p75^(NTR) ICD sites required forthe interaction with Rab5 and Rab31. Schematic diagram of the p75^(NTR)ICD shows the domain organization. The six helixes within the DD arehighlighted in yellow. Peptide array screened with recombinant GST-Rab5and GST-Rab31 revealed helix 4 of p75^(NTR) ICD as the strongest regionthat interacts with Rab5 and Rab31. Peptide location, length, andsequences are shown. Peptide library was also screened with recombinantGST as a control. (I) Co-immunoprecipitation of p75ICD and activatedGST-Rab5 recombinant proteins. Tat-Pep5 or Tat control peptide(Tat-ctrl) were added as indicated. GST-Rab5 was immunoprecipitated withanti-GST antibody, and western blots were developed with anti-p75^(NTR)and anti-GST antibodies. Western blots were performed in triplicates andin all panels representative blots are shown.

FIG. 10.

(A) Immunoprecipitation of myc-GAPEX-5 with HA-FL-p75^(NTR) transfectedin HEK293T cells. Lysates were immunoprecipitated with anti-mycantibody, and western blots were developed with anti-HA and anti-mycantibodies. (B) Immunolocalization of p75^(NTR) with Rab31 or Rab5 in3T3L1 adipocytes. Differentiated 3T3L1 were fixed and immunostained forRab31 and Rab5 with p75^(NTR). Images show co-immunolocalization ofRab31 and Rab5, respectively with p75^(NTR). (C) Cytosolic (CYT) andlow-density microsome (LDM) fractions of 3T3L1 adipocytes show presenceof p75^(NTR) and Rab5 in LDMs. Caveolin was used as a membrane fractionmarker. (D) Schematic representation of the binding of Rab5 familyGTPases and RhoGDI to helixes 4 and 5 of the death domain of p75^(NTR)respectively.

Example 2 Identification of Rab31 Peptides that Bind p75^(NTR) ICD

Peptide arrays were manufactured by Kinexus Bioinformatics Corporation(Vancouver, Canada). Synthetic overlapping peptides (25 amino acids inlength) for Rab31 were spotted on nitrocellulose membranes. Membraneswere overlaid with 1 μg/ml recombinant GST-p75^(NTR) ICD. Boundrecombinant GST-p75ICD were detected using rabbit anti-p75^(NTR)(1:1000, Millipore) followed by secondary anti-rabbit horseradishperoxidase antibody (1:5000, Cell Signaling).

As shown in FIG. 16, Peptide 6 was identified as a Rab31 peptide thatbinds p75^(NTR) ICD.

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While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of reducing blood glucose levels and/orinsulin resistance in an individual, the method comprising administeringto the individual an effective amount of an agent that inhibits bindingbetween the intracellular domain of the p75 neurotrophin receptor(p75^(NTR)) and an intracellular GTPase selected from Rab31 and Rab5,wherein the agent is a peptide comprising an amino acid sequenceselected from: a) LLASWATQDSATLDA; (SEQ ID NO: 1) b) X₁LASWATQDSATLDA;(SEQ ID NO: 2) c) LX₁ASWATQDSATLDA; (SEQ ID NO: 3) d) LLX₁SWATQDSATLDA;(SEQ ID NO: 4) e) LLAX₁WATQDSATLDA; (SEQ ID NO: 5) f) LLASX₁ATQDSATLDA;(SEQ ID NO: 6) g) LLASWX₁TQDSATLDA; (SEQ ID NO: 7) h) LLASWAX₁QDSATLDA;(SEQ ID NO: 8) i) LLASWATX₁DSATLDA; (SEQ ID NO: 9) j) LLASWATQX₁SATLDA;(SEQ ID NO: 10) k) LLASWATQDX₁ATLDA; (SEQ ID NO: 11) l)LLASWATQDSX₁TLDA; (SEQ ID NO: 12) m) LLASWATQDSAX₁LDA; (SEQ ID NO: 13)n) LLASWATQDSATX₁DA; (SEQ ID NO: 14) o) LLASWATQDSATLX₁A;(SEQ ID NO: 15) and p) LLASWATQDSATLDX₁, (SEQ ID NO: 16)

where X₁ is any amino acid.
 2. The method of claim 1, wherein thepeptide comprises at least one non-coded amino acid.
 3. The method ofclaim 1, wherein the peptide comprises at least one D-amino acid.
 4. Themethod of claim 1, wherein the peptide comprises a non-peptide isostericlinkage.
 5. The method of claim 1, wherein the peptide has a length offrom 15 amino acids to about 50 amino acids.
 6. The method of claim 1,wherein the peptide is cyclized.
 7. The method of claim 1, wherein thepeptide comprises a protein transduction domain (PTD).
 8. The method ofclaim 1, wherein the agent is a peptide comprising the amino acidsequence LLASWATQDSATLDA (SEQ ID NO:1).